Cells within a tissue of a subject have differences in cell morphology and/or function due to varied analyte levels (e.g., gene and/or protein expression) within the different cells. The specific position of a cell within a tissue (e.g., the cell's position relative to neighboring cells or the cell's position relative to the tissue microenvironment) can affect, e.g., the cell's morphology, differentiation, fate, viability, proliferation, behavior, and signaling and cross-talk with other cells in the tissue.
Spatial heterogeneity has been previously studied using techniques that only provide data for a small handful of analytes in the context of an intact tissue or a portion of a tissue, or provide a lot of analyte data for single cells, but fail to provide information regarding the position of the single cell in a parent biological sample (e.g., tissue sample).
Spatial analysis takes advantage of targeting a particular analyte in a sample using a capture probe that includes a capture domain capable of capturing the particular analyte. In the context of RNA, one approach developed to enhance resolution of spatial analysis is a process called RNA-templated ligation (RTL), which includes multiple oligonucleotides targeting adjacent or nearby complementary sequences on the analyte (e.g., RNA). RTL methods have not been adapted to different types of nucleic acid analytes in the context of spatial analysis. Thus, there remains a need to adapt RTL methods to the spatial analysis of a genomic DNA (gDNA) molecule.
Featured herein are methods of identifying a target gDNA analyte of interest or a genetic variant in a gDNA analyte. In particular, methods provided herein co-opt traditional RTL principles to include oligonucleotide probes that hybridize to adjacent complementary genomic DNA (gDNA) sequences. Methods provided herein allow a readout of a DNA-templated hybridization event that does not require single stranded DNA from the DNA-DNA template. For example, an oligonucleotide (e.g., a second RTL probe) that hybridizes to the gDNA analyte includes a 5′ FLAP sequence that can be removed and detected upon an enzyme-mediated cleavage event that does not require removal of the hybridized oligonucleotide probes. Detection of the 5′ FLAP provides a readout for the DNA-templated hybridization event, which can serve as a proxy for detecting a gDNA analyte or a genetic variant within a gDNA analyte.
In one aspect, the disclosure features a method for determining the abundance and/or location of a genomic DNA (gDNA) analyte in a biological sample, the method comprising: (a) contacting the biological sample with a substrate comprising a plurality of capture probes, wherein a capture probe of the plurality comprises a spatial barcode and a capture domain; (b) contacting the biological sample with a first RTL probe and a second RTL probe, wherein the first RTL probe and the second RTL probe are substantially complementary to adjacent sequences of the gDNA analyte, and wherein the second RTL probe comprises a 5′ FLAP; (c) hybridizing the first RTL probe and the second RTL probe to the gDNA analyte; (d) cleaving the second RTL probe, thereby releasing the 5′ FLAP; (e) hybridizing the 5′ FLAP to the capture domain; and (f) determining (i) all or a part of the sequence of the 5′ FLAP, or a complement thereof, and (ii) all or a part of the sequence of the spatial barcode, or a complement thereof, and using the determined sequence of (i) and (ii) to identify the abundance and/or location of the gDNA analyte in the biological sample.
In some embodiments, the first RTL probe and the second RTL probes are DNA probes.
In some embodiments, the 5′ FLAP comprises a first barcode sequence, wherein the first barcode sequence comprises a sequence that identifies the first RTL probe, the second RTL probe, the gDNA analyte, or any combination thereof.
In some embodiments, the 5′ FLAP comprises (i) a functional sequence, wherein the functional sequence is a primer sequence, and (ii) a capture probe binding domain, wherein the capture probe binding domain comprises a homopolymeric sequence or a poly(A) sequence.
In some embodiments, the cleaving the second RTL probe comprises providing an endonuclease, wherein the endonuclease cleaves a portion of the first RTL probe, a portion of second RTL probe, the 5′ FLAP of the second RTL probe, or any combination thereof.
In some embodiments, the determining step comprises sequencing all or part of the 5′ FLAP.
In some embodiments, the biological sample is a formalin-fixed, paraffin-embedded (FFPE) sample.
In another aspect, the disclosure features a method of detecting a genetic variant in a gDNA analyte in a biological sample, the method comprising: (a) contacting the biological sample with a substrate comprising a plurality of capture probes, wherein a capture probe of the plurality comprises a spatial barcode and a capture domain; (b) contacting the biological sample with a first RTL probe and a second RTL probe, wherein the first RTL probe and the second RTL probe each comprise a sequence that is substantially complementary to adjacent sequences of the gDNA analyte; wherein the first RTL probe and the second RTL probe are capable of forming an invasive cleavage structure in the presence of the genetic variant; and wherein the second RTL probe further comprises a 5′ FLAP; (c) hybridizing the first RTL probe and the second RTL probe to the gDNA analyte; (d) cleaving the second RTL probe when the genetic variant is present, thereby releasing the 5′ FLAP; (e) hybridizing the 5′ FLAP to the capture domain; and (f) determining (i) all or a part of the sequence of the 5′ FLAP, or a complement thereof, and (ii) all or a part of the sequence of the spatial barcode, or a complement thereof, and using the determined sequence of (i) and (ii) to detect the genetic variant in the gDNA analyte in the biological sample.
In some embodiments, the genetic variant is a single nucleotide polymorphism (SNP) or a nucleotide point mutation, or wherein the genetic variant comprises at least two, at least three, at least four, at least five, or more genetic variants.
In some embodiments, the first RTL probe comprises a sequence that is substantially complementary to a sequence 3′ to the genetic variant, or at least one nucleotide that is complementary to a wild-type sequence of the genetic variant.
In some embodiments, the second RTL probe comprises a sequence substantially complementary to a sequence 5′ to the genetic variant, and/or a nucleotide that is complementary to the genetic variant.
In some embodiments, the 5′ FLAP comprises a nucleotide that is complementary to the genetic variant.
In some embodiments, the first RTL probe and the second RTL probe are capable of forming an invasive cleavage structure in the presence of the genetic variant.
In some embodiments, the 5′ FLAP comprises a capture probe binding domain, wherein the capture probe binding domain comprises a homopolymeric sequence or a poly(A) sequence.
In some embodiments, the 5′ FLAP comprises from 5′ to 3′; a functional sequence, a barcode, and a capture probe binding domain sequence.
In some embodiments, the 5′ FLAP comprises from 5′ to 3′; a functional sequence, a barcode, a capture probe binding domain sequence, and an additional nucleotide, wherein the additional nucleotide comprises a nucleotide that is complementary to the genetic variant or a nucleotide that is complementary to the wild type sequence of the genetic variant.
In some embodiments, the method further comprises providing a capture probe binding domain blocking moiety that interacts with the capture probe binding domain, wherein the method further comprises releasing the capture probe binding domain blocking moiety from the capture probe binding domain prior to contacting the biological sample with the substrate, wherein the capture probe binding domain blocking moiety comprises a poly-uridine sequence, a poly-thymidine sequence, or both, and wherein releasing the capture probe binding domain blocking moiety from the poly(A) sequence comprises denaturing the ligated probe.
In some embodiments, the biological sample is an FFPE sample.
In another aspect, the disclosure features a kit comprising: (a) a first RTL probe and a second RTL probe, wherein the first RTL probe and the second RTL probe are substantially complementary to adjacent sequences of a gDNA analyte, or wherein the first RTL probe and the second RTL probe each comprise a sequence that is substantially complementary to adjacent sequences of the gDNA analyte, and wherein the second RTL probe comprises a 5′ FLAP; (b) an endonuclease, wherein the endonuclease cleaves the 5′ FLAP thereby releasing the 5′ FLAP from the second RTL probe; (c) a substrate comprising a plurality of capture probes, wherein a capture probe of the plurality comprises a spatial barcode and a capture domain; and (d) instructions for performing any one of the methods described herein.
In another aspect, the disclosure features a composition for determining the abundance and/or location of a gDNA analyte in a biological sample, wherein the composition comprises: a first RTL probe and a second RTL probe hybridized to the gDNA analyte, wherein the first RTL probe and the second RTL probe are substantially complementary to adjacent sequences of the gDNA analyte, or wherein the first RTL probe and the second RTL probe each comprise a sequence that is substantially complementary to adjacent sequences of the gDNA analyte, and wherein the second RTL probe comprises a 5′ FLAP, wherein the 5′ FLAP comprises a sequence that is capable of hybridizing to a capture domain of a capture probe.
In one aspect, this disclosure features methods for detecting a genomic DNA (gDNA) molecule in a biological sample, including: (a) contacting the biological sample with a first probe oligonucleotide and a second probe oligonucleotide, wherein the first probe oligonucleotide and the second probe oligonucleotide are substantially complementary to adjacent sequences of the gDNA molecule, and wherein the second probe oligonucleotide includes a 5′ FLAP; (b) hybridizing the first probe oligonucleotide and the second probe oligonucleotide to the gDNA molecule; (c) cleaving the second probe oligonucleotide, thereby releasing the 5′ FLAP; and (d) determining all or a part of the sequence of the 5′ FLAP to detect the gDNA molecule.
In another aspect, this disclosure features methods of detecting a genetic variant in a gDNA molecule in a biological sample including: (a) contacting the biological sample with a first probe oligonucleotide and a second probe oligonucleotide, wherein the first probe oligonucleotide and the second probe oligonucleotide each include a sequence that is substantially complementary to adjacent sequences of the gDNA molecule; wherein the first probe oligonucleotide and the second probe oligonucleotide are capable of forming an invasive cleavage structure in the presence of the genetic variant; and wherein the second probe oligonucleotide further includes a 5′ FLAP; (b) hybridizing the first probe oligonucleotide and the second probe oligonucleotide to the gDNA molecule; (c) cleaving the second probe oligonucleotide when the genetic variant is present, thereby releasing the 5′ FLAP; and (d) determining all or a part of the sequence of the 5′ FLAP to detect the genetic variant in the gDNA molecule.
In some embodiments, the gDNA molecule includes one or more single nucleotide variants compared to a reference gDNA molecule.
In some embodiments, the first probe oligonucleotide includes a sequence that is substantially complementary to a sequence 3′ to the genetic variant. In some embodiments, the first probe oligonucleotide further includes at least one nucleotide that is complementary to a wild-type sequence of the genetic variant. In some embodiments, the first probe oligonucleotide includes at least two ribonucleic acid bases at the 3′ end. In some embodiments, the first probe oligonucleotide includes at least two deoxyribonucleic acid bases at the 3′ end.
In some embodiments, the second probe oligonucleotide includes a sequence substantially complementary to a sequence 5′ to the genetic variant.
In some embodiments, the 5′ FLAP further includes a first barcode sequence. In some embodiments, the first barcode sequence includes a sequence that identifies at least one of the first probe oligonucleotide, the second probe oligonucleotide, or the gDNA molecule.
In some embodiments, the 5′ FLAP further includes a functional sequence. In some embodiments, the functional sequence is selected from a primer sequence, a Read 1 sequence, a Read 2 sequence, an index sequence, a P5 index sequence, and a P7 index sequence.
In some embodiments, the 5′ FLAP further includes a nucleotide that is complementary to the genetic variant. In some embodiments, the second probe oligonucleotide further includes a second barcode.
In some embodiments, the second probe oligonucleotide includes a sequence that is complementary to a capture domain. In some embodiments, the second probe oligonucleotide further includes a nucleotide that is complementary to the genetic variant.
In some embodiments, the cleaving step includes providing an endonuclease. In some embodiments, the endonuclease cleaves the invasive cleavage structure.
In some embodiments, the endonuclease cleaves a portion of the first probe oligonucleotide. In some embodiments, the endonuclease cleaves the at least one nucleotide that is complementary to a wild-type sequence of the gDNA molecule of the first probe oligonucleotide. In some embodiments, the endonuclease cleaves a portion of second probe oligonucleotide. In some embodiments, the endonuclease cleaves the 5′ FLAP of the second probe oligonucleotide. In some embodiments, the endonuclease is FLAP endonuclease 1 (FEN1).
In some embodiments, the method further includes contacting the biological sample with a permeabilization reagent.
In some embodiments, the biological sample is contacted with the permeabilization reagent before step (d) (e.g., determining all or a part of the sequence of the 5′ FLAP to detect the gDNA molecule).
In some embodiments, the method further includes denaturing the gDNA under conditions wherein the first probe oligonucleotide and the second probe oligonucleotide can hybridize to the gDNA molecule.
In some embodiments, the determining step includes amplifying all or part of the 5′ FLAP. In some embodiments, the amplifying is isothermal. In some embodiments, the amplifying is not isothermal. In some embodiments, the determining step includes sequencing.
In another aspect, this disclosure features methods for detecting a gDNA molecule at a spatial location in a biological sample, including: (a) contacting a biological sample with a first probe oligonucleotide and a second probe oligonucleotide, wherein the first probe oligonucleotide and the second probe oligonucleotide are substantially complementary to adjacent sequences of the gDNA molecule, and wherein the second probe oligonucleotide includes a 5′ FLAP; (b) hybridizing the first probe oligonucleotide and the second probe oligonucleotide to the gDNA molecule; (c) cleaving the second probe oligonucleotide, thereby releasing the 5′ FLAP; (d) contacting the biological sample with a substrate including a plurality of capture probes, wherein a capture probe of the plurality includes a spatial barcode and a capture domain; (e) hybridizing the 5′ FLAP to the capture domain; and (f) determining (i) all or a part of the sequence of the 5′ FLAP, or a complement thereof, and (ii) all or a part of the sequence of the spatial barcode, or a complement thereof, and using the determined sequence of (i) and (ii) to identify the spatial location of the gDNA molecule in the biological sample.
In another aspect, this disclosure features methods of detecting a genetic variant in a genomic DNA (gDNA) molecule at a spatial location in a biological sample including: (a) contacting the biological sample with a first probe oligonucleotide and a second probe oligonucleotide, wherein the first probe oligonucleotide and the second probe oligonucleotide each include a sequence that is substantially complementary to adjacent sequences of the gDNA molecule; wherein the first probe oligonucleotide and the second probe oligonucleotide are capable of forming an invasive cleavage structure in the presence of the genetic variant; and wherein the second probe oligonucleotide further includes a 5′ FLAP; (b) hybridizing the first probe oligonucleotide and the second probe oligonucleotide to the gDNA molecule; (c) cleaving the second probe oligonucleotide when the genetic variant is present, thereby releasing the 5′ FLAP; (d) contacting the biological sample with a substrate including a plurality of capture probes, wherein a capture probe of the plurality includes a spatial barcode and a capture domain; (e) hybridizing the cleavage product to the capture domain; and (f) determining (i) all or a part of the sequence of the 5′ FLAP, or a complement thereof, and (ii) all or a part of the sequence of the spatial barcode, or a complement thereof, and using the determined sequence of (i) and (ii) to identify the spatial location of the gDNA molecule in the biological sample.
In some embodiments, the gDNA molecule includes one or more single nucleotide variants compared to a reference gDNA molecule.
In some embodiments, the first probe oligonucleotide includes a sequence that is substantially complementary to a sequence 3′ of the genetic variant. In some embodiments, the first probe oligonucleotide further includes at least one nucleotide that is complementary to a wild-type sequence of the genetic variant. In some embodiments, the first probe oligonucleotide includes at least two ribonucleic acid bases at the 3′ end. In some embodiments, the first probe oligonucleotide includes at least two deoxyribonucleic acid bases at the 3′ end.
In some embodiments, the second probe oligonucleotide includes a sequence substantially complementary to a sequence 5′ to the genetic variant.
In some embodiments, the 5′ FLAP further includes a first barcode sequence. In some embodiments, the first barcode sequence includes a sequence that identifies at least one of the first probe oligonucleotide, the second probe oligonucleotide, or the gDNA molecule. In some embodiments, the first barcode sequence includes a sequence that identifies the gDNA molecule.
In some embodiments, the 5′ FLAP further includes a functional sequence. In some embodiments, the functional sequence is selected from a primer sequence, a Read 1 sequence, a Read 2 sequence, an index sequence, a P5 index sequence, and a P7 index sequence.
In some embodiments, the 5′ FLAP further includes a capture probe binding domain. In some embodiments, the capture probe binding domain includes a homopolymeric sequence. In some embodiments, the capture probe binding domain includes a poly(A) sequence. In some embodiments, the 5′ FLAP further includes a nucleotide that is complementary to the genetic variant.
In some embodiments, the 5′ FLAP includes from 5′ to 3′; a functional sequence, a barcode, and a capture probe binding domain sequence. In some embodiments, the 5′ FLAP includes from 5′ to 3′; a functional sequence, a barcode, a capture probe binding domain sequence, and an additional nucleotide. In some embodiments, the additional nucleotide includes a nucleotide that is complementary to the genetic variant. In some embodiments, the additional nucleotide includes a nucleotide that is complementary to the wild type sequence of the genetic variant.
In some embodiments, the method further includes providing a capture probe binding domain blocking moiety that interacts with the capture probe binding domain.
In some embodiments, the method further includes releasing the capture probe binding domain blocking moiety from the capture probe binding domain prior to contacting the biological sample with the substrate. In some embodiments, the capture probe binding domain blocking moiety includes a poly-uridine sequence, a poly-thymidine sequence, or both. In some embodiments, releasing the capture probe binding domain blocking moiety from the poly(A) sequence includes denaturing the ligated probe.
In some embodiments, the second probe oligonucleotide further includes a second barcode.
In some embodiments, the second probe oligonucleotide further includes a second capture probe binding domain.
In some embodiments, the second probe oligonucleotide further includes a nucleotide that is complementary to the genetic variant.
In some embodiments, the cleaving step includes providing an endonuclease. In some embodiments, the endonuclease cleaves the invasive cleavage structure. In some embodiments, the endonuclease cleaves a portion of the first probe oligonucleotide. In some embodiments, the endonuclease cleaves the at least one nucleotide that is complementary to a wild-type sequence of the gDNA molecule of the first probe oligonucleotide. In some embodiments, the endonuclease cleaves a portion of second probe oligonucleotide. In some embodiments, the endonuclease cleaves the 5′ FLAP of the second probe oligonucleotide. In some embodiments, the endonuclease is FLAP endonuclease 1 (FEN1).
In some embodiments, the method further includes contacting the biological sample with a permeabilization reagent. In some embodiments, the biological sample is contacted with the permeabilization reagent before step (d) (e.g., contacting the biological sample with a substrate including a plurality of capture).
In some embodiments, the method further includes denaturing the gDNA under conditions wherein the first probe oligonucleotide and the second probe oligonucleotide can hybridize to the gDNA molecule.
In some embodiments, the determining step includes amplifying all or part of the 5′ FLAP specifically bound to the capture domain. In some embodiments, the amplifying is isothermal. In some embodiments, the amplifying is not isothermal. In some embodiments, the determining step includes sequencing.
In some embodiments of any of the methods for detecting a genetic variant in a gDNA molecule, the genetic variant is a single nucleotide polymorphism (SNP). In some embodiments of any of the methods for detecting a genetic variant in a gDNA molecule, the genetic variant is a nucleotide point mutation. In some embodiments of any of the methods for detecting a genetic variant in a gDNA molecule, the genetic variant includes at least two, at least three, at least four, at least five, or more genetic variants.
In some embodiments, the first probe oligonucleotide is at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% complementary to a sequence of the gDNA molecule. In some embodiments, the second probe oligonucleotide is at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% complementary to a sequence of the gDNA molecule.
In some embodiments, the biological sample is a formalin-fixed, paraffin-embedded (FFPE), frozen, or fresh sample. In some embodiments, the biological sample is a FFPE sample.
In another aspect, this disclosure features a kit for use in a method of detecting analyte in a biological sample, wherein the kit includes any two or more of: (a) a first probe oligonucleotide and a second probe oligonucleotide, wherein the first probe oligonucleotide and the second probe oligonucleotide are substantially complementary to adjacent sequences of the analyte, and wherein the second probe oligonucleotide includes a 5′ FLAP; (b) an endonuclease, wherein the endonuclease cleaves the 5′ FLAP thereby releasing the 5′ FLAP from the second probe oligonucleotide; (c) a substrate including a plurality of capture probes, wherein a capture probe of the plurality includes a spatial barcode and a capture domain, wherein the 5′ FLAP is capable of hybridizing to the capture domain; and (d) instructions for carrying out any of the methods described herein.
In another aspect, this disclosure features a kit for use in a method of detecting analyte in a biological sample, wherein the kit includes any two or more of: (a) a first probe oligonucleotide and a second probe oligonucleotide, wherein the first probe oligonucleotide and the second probe oligonucleotide are substantially complementary to adjacent sequences of the analyte, and wherein the second probe oligonucleotide includes a 5′ FLAP; (b) an endonuclease, wherein the endonuclease cleaves the 5′ FLAP thereby releasing the 5′ FLAP from the second probe oligonucleotide; and (c) instructions for carrying out any of the methods described herein.
In another aspect, this disclosure features a kit for use in a method of detecting a genetic variant in an gDNA molecule in a biological sample, wherein the kit includes any two or more of: (a) a first probe oligonucleotide and a second probe oligonucleotide, wherein the first probe oligonucleotide and the second probe oligonucleotide each include a sequence that is substantially complementary to adjacent sequences of the gDNA molecule; wherein the first probe oligonucleotide and the second probe oligonucleotide are capable of forming an invasive cleavage structure in the presence of the genetic variant; and wherein the second probe oligonucleotide further includes a 5′ FLAP; (b) an endonuclease, wherein the endonuclease cleaves the 5′ FLAP thereby releasing the 5′ FLAP from the second probe oligonucleotide; (c) a substrate including a plurality of capture probes, wherein a capture probe of the plurality includes a spatial barcode and a capture domain, wherein the 5′ FLAP is capable of hybridizing to the capture domain; and (d) instructions for carrying out any of the methods described herein.
In another aspect, this disclosure features a kit for use in a method of detecting a genetic variant in an gDNA molecule in a biological sample, wherein the kit includes any two or more of: (a) a first probe oligonucleotide and a second probe oligonucleotide, wherein the first probe oligonucleotide and the second probe oligonucleotide each include a sequence that is substantially complementary to adjacent sequences of the gDNA molecule; wherein the first probe oligonucleotide and the second probe oligonucleotide are capable of forming an invasive cleavage structure in the presence of the genetic variant; and wherein the second probe oligonucleotide further includes a 5′ FLAP; (b) an endonuclease, wherein the endonuclease cleaves the 5′ FLAP thereby releasing the 5′ FLAP from the second probe oligonucleotide; and (c) instructions for carrying out any of the methods described herein.
In another aspect, this disclosure features compositions including: a first probe oligonucleotide and a second probe oligonucleotide hybridized to an analyte, wherein the first probe oligonucleotide and the second probe oligonucleotide are substantially complementary to adjacent sequences of the analyte, and wherein the second probe oligonucleotide includes a 5′ FLAP.
In another aspect, this disclosure features compositions including: a first probe oligonucleotide and a second probe oligonucleotide hybridized to an analyte, wherein the first probe oligonucleotide and the second probe oligonucleotide each include a sequence that is substantially complementary to adjacent sequences of the gDNA molecule; wherein the first probe oligonucleotide and the second probe oligonucleotide are capable of forming an invasive cleavage structure in the presence of the genetic variant; and wherein the second probe oligonucleotide further includes a 5′ FLAP. In some embodiments, the 5′ FLAP includes a sequence that is capable of hybridizing to a capture domain of a capture probe. In some embodiments, the capture probe further includes a spatial barcode. In some embodiments, the capture probe is part of a plurality of capture probes affixed to a substrate.
All publications, patents, patent applications, and information available on the internet and 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.
The singular form “a”, “an”, and “the” include plural references unless the context clearly dictates otherwise. For example, the term “a cell” includes one or more cells, comprising mixtures thereof. “A and/or B” is used herein to include all of the following alternatives: “A”, “B”, “A or B”, and “A and B”.
Various embodiments of the features of this disclosure are described herein. However, it should be understood that such embodiments are provided merely by way of example, and numerous variations, changes, and substitutions can occur to those skilled in the art without departing from the scope of this disclosure. It should also be understood that various alternatives to the specific embodiments described herein are also within the scope of this disclosure.
The following drawings illustrate certain embodiments of the features and advantages of this disclosure. These embodiments are not intended to limit the scope of the appended claims in any manner. Like reference symbols in the drawings indicate like elements.
I. Methods and Compositions for Spatial Analysis
Spatial analysis methodologies and compositions described herein can provide a vast amount of analyte and/or expression data for a variety of analytes within a biological sample at high spatial resolution, while retaining native spatial context. Spatial analysis methods and compositions can include, e.g., the use of a capture probe including a spatial barcode (e.g., a nucleic acid sequence that provides information as to the location or position of an analyte within a cell or a tissue sample (e.g., mammalian cell or a mammalian tissue sample) and a capture domain that is capable of binding to an analyte (e.g., a protein and/or a nucleic acid) produced by and/or present in a cell. Spatial analysis methods and compositions can also include the use of a capture probe having a capture domain that captures an intermediate agent for indirect detection of an analyte. For example, the intermediate agent can include a nucleic acid sequence (e.g., a barcode) associated with the intermediate agent. Detection of the intermediate agent is therefore indicative of the analyte in the cell or tissue sample.
Non-limiting aspects of spatial analysis methodologies and compositions are described in U.S. Pat. Nos. 10,774,374, 10,724,078, 10,480,022, 10,059,990, 10,041,949, 10,002,316, 9,879,313, 9,783,841, 9,727,810, 9,593,365, 8,951,726, 8,604,182, 7,709,198, U.S. Patent Application Publication Nos. 2020/239946, 2020/080136, 2020/0277663, 2020/024641, 2019/330617, 2019/264268, 2020/256867, 2020/224244, 2019/194709, 2019/161796, 2019/085383, 2019/055594, 2018/216161, 2018/051322, 2018/0245142, 2017/241911, 2017/089811, 2017/067096, 2017/029875, 2017/0016053, 2016/108458, 2015/000854, 2013/171621, WO 2018/091676, WO 2020/176788, 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 C, dated June 2020), and/or the Visium Spatial Tissue Optimization Reagent Kits User Guide (e.g., Rev C, dated July 2020), both of which are available at the 10× Genomics Support Documentation website, and can be used herein in any combination. Further non-limiting aspects of spatial analysis methodologies and compositions are described herein.
Some general terminology that may be used in this disclosure can be found in Section (I)(b) of WO 2020/176788 and/or U.S. Patent Application Publication No. 2020/0277663. Typically, a “barcode” is a label, or identifier, that conveys or is capable of conveying information (e.g., information about an analyte in a sample, a bead, and/or a capture probe). A barcode can be part of an analyte, or independent of an analyte. A barcode can be attached to an analyte. A particular barcode can be unique relative to other barcodes. For the purpose of this disclosure, an “analyte” can include any biological substance, structure, moiety, or component to be analyzed. The term “target” can similarly refer to an analyte of interest.
Analytes can be broadly classified into one of two groups: nucleic acid analytes, and non-nucleic acid analytes. Examples of non-nucleic acid analytes include, but are not limited to, lipids, carbohydrates, peptides, proteins, glycoproteins (N-linked or O-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 WO 2020/176788 and/or U.S. Patent Application Publication No. 2020/0277663. In some embodiments, an analyte can be detected indirectly, such as through detection of an intermediate agent, for example, a ligation product or an analyte capture agent (e.g., an oligonucleotide-conjugated antibody), such as those described herein.
A “biological sample” is typically obtained from the subject for analysis using any of a variety of techniques including, but not limited to, biopsy, surgery, and laser capture microscopy (LCM), and generally includes cells and/or other biological material from the subject. In some embodiments, a biological sample can be a tissue section. In some embodiments, a biological sample can be a fixed and/or stained biological sample (e.g., a fixed and/or stained tissue section). Non-limiting examples of stains include histological stains (e.g., hematoxylin and/or eosin) and immunological stains (e.g., fluorescent stains). In some embodiments, a biological sample (e.g., a fixed and/or stained biological sample) can be imaged. Biological samples are also described in Section (I)(d) of WO 2020/176788 and/or U.S. Patent Application Publication No. 2020/0277663.
In some embodiments, a biological sample is 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 WO 2020/176788 and/or U.S. Patent Application Publication No. 2020/0277663.
Array-based spatial analysis methods involve the transfer of one or more analytes from a biological sample to an array of features on a substrate, where each feature is associated with a unique spatial location on the array. Subsequent analysis of the transferred analytes includes determining the identity of the analytes and the spatial location of the analytes within the biological sample. The spatial location of an analyte within the biological sample is determined based on the feature to which the analyte is bound (e.g., directly or indirectly) on the array, and the feature's relative spatial location within the array.
A “capture probe” refers to any molecule capable of capturing (directly or indirectly) and/or labelling an analyte (e.g., an analyte of interest) in a biological sample. In some embodiments, the capture probe is a nucleic acid or a polypeptide. In some embodiments, the capture probe includes a barcode (e.g., a spatial barcode and/or a unique molecular identifier (UMI)) and a capture domain). In some embodiments, a capture probe can include a cleavage domain and/or a functional domain (e.g., a primer-binding site, such as for next-generation sequencing (NGS)). See, e.g., Section (II)(b) (e.g., subsections (i)-(vi)) of WO 2020/176788 and/or U.S. Patent Application Publication No. 2020/0277663. Generation of capture probes can be achieved by any appropriate method, including those described in Section (II)(d)(ii) of WO 2020/176788 and/or U.S. Patent Application Publication No. 2020/0277663.
In some embodiments, the capture probe further includes a unique molecular identifier. In some embodiments, the capture probe further includes a functional domain. In some embodiments, the capture domain includes a sequence that is partially complementary to the analyte or an analyte binding moiety. In some embodiments, the capture domain includes a homopolymeric sequence. In some embodiments, the capture domain includes a poly(T) sequence. In some embodiments, the capture probe further includes a cleavage domain. In some embodiments, the cleavage domain includes a cleavable linker. Non-limiting examples of a cleavable linker include a photocleavable linker, a UV-cleavable linker, an enzymatic cleavable linker, or a pH-sensitive cleavable linker. In some embodiments, the plurality of capture probes are attached to one or more features.
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 105 and functional sequences 104 is common to all of the probes attached to a given feature. In some embodiments, the UMI sequence 106 of a capture probe attached to a given feature is different from the UMI sequence of a different capture probe attached to the given feature.
For multiple capture probes that are attached to a common array feature, the one or more spatial barcode sequences of the multiple capture probes can include sequences that are the same for all capture probes coupled to the feature, and/or sequences that are different across all capture probes coupled to the feature.
Capture probes attached to a single array feature can include identical (or common) spatial barcode sequences, different spatial barcode sequences, or a combination of both. Capture probes attached to a feature can include multiple sets of capture probes. Capture probes of a given set can include identical spatial barcode sequences. The identical spatial barcode sequences can be different from spatial barcode sequences of capture probes of another set.
The plurality of capture probes can include spatial barcode sequences (e.g., nucleic acid barcode sequences) that are associated with specific locations on a spatial array. For example, a first plurality of capture probes can be associated with a first region, based on a spatial barcode sequence common to the capture probes within the first region, and a second plurality of capture probes can be associated with a second region, based on a spatial barcode sequence common to the capture probes within the second region. The second region may or may not be associated with the first region. Additional pluralities of capture probes can be associated with spatial barcode sequences common to the capture probes within other regions. In some embodiments, the spatial barcode sequences can be the same across a plurality of capture probe molecules.
In some embodiments, multiple different spatial barcodes are incorporated into a single arrayed capture probe. For example, a mixed but known set of spatial barcode sequences can provide a stronger address or attribution of the spatial barcodes to a given spot or location, by providing duplicate or independent confirmation of the identity of the location. In some embodiments, the multiple spatial barcodes represent increasing specificity of the location of the particular array point.
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 WO 2020/176788 and/or U.S. Patent Application Publication No. 2020/0277663.
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 or capture handle 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” or “capture handle 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 embodiments, a capture handle sequence is complementary to 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 WO 2020/176788 and/or Section (II)(b)(viii) U.S. Patent Application Publication No. 2020/0277663.
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 template, such as an analyte or an intermediate agent (e.g., a ligation product or an analyte capture agent), or a portion thereof), or derivatives thereof (see, e.g., Section (II)(b)(vii) of WO 2020/176788 and/or U.S. Patent Application Publication No. 2020/0277663 regarding extended capture probes). In some cases, capture probes may be configured to form ligation products with a template (e.g., a DNA template, such as an analyte or an intermediate agent, or portion thereof), thereby creating ligations products that serve as proxies for a template.
As used herein, an “extended capture probe” refers to a capture probe having additional nucleotides added to the terminus (e.g., 3′ or 5′ end) of the capture probe thereby extending the overall length of the capture probe. For example, an “extended 3′ end” indicates additional nucleotides were added to the most 3′ nucleotide of the capture probe to extend the length of the capture probe, for example, by polymerization reactions used to extend nucleic acid molecules including templated polymerization catalyzed by a polymerase (e.g., a DNA polymerase or a reverse transcriptase). In some embodiments, extending the capture probe includes adding to a 3′ end of a capture probe a nucleic acid sequence that is complementary to a nucleic acid sequence of an analyte or intermediate agent specifically bound to the capture domain of the capture probe. In some embodiments, the capture probe is extended using reverse transcription. In some embodiments, the capture probe is extended using one or more DNA polymerases. The extended capture probes include the sequence of the capture probe and the sequence of the spatial barcode of the capture probe.
In some embodiments, extended capture probes are amplified (e.g., in bulk solution or on the array) to yield quantities that are sufficient for downstream analysis, e.g., via DNA sequencing. In some embodiments, extended capture probes (e.g., DNA molecules) act as templates for an amplification reaction (e.g., a polymerase chain reaction).
Additional variants of spatial analysis methods, including in some embodiments, an imaging step, are described in Section (II)(a) of WO 2020/176788 and/or U.S. Patent Application Publication No. 2020/0277663. Analysis of captured analytes (and/or intermediate agents or portions thereof), for example, including sample removal, extension of capture probes, sequencing (e.g., of a cleaved extended capture probe and/or a cDNA molecule complementary to an extended capture probe), sequencing on the array (e.g., using, for example, in situ hybridization or in situ ligation approaches), temporal analysis, and/or proximity capture, is described in Section (II)(g) of WO 2020/176788 and/or U.S. Patent Application Publication No. 2020/0277663. Some quality control measures are described in Section (II)(h) of WO 2020/176788 and/or U.S. Patent Application Publication No. 2020/0277663.
Spatial information can provide information of biological and/or medical importance. For example, the methods and compositions described herein can allow for: identification of one or more biomarkers (e.g., diagnostic, prognostic, and/or for determination of efficacy of a treatment) of a disease or disorder; identification of a candidate drug target for treatment of a disease or disorder; identification (e.g., diagnosis) of a subject as having a disease or disorder; identification of stage and/or prognosis of a disease or disorder in a subject; identification of a subject as having an increased likelihood of developing a disease or disorder; monitoring of progression of a disease or disorder in a subject; determination of efficacy of a treatment of a disease or disorder in a subject; identification of a patient subpopulation for which a treatment is effective for a disease or disorder; modification of a treatment of a subject with a disease or disorder; selection of a subject for participation in a clinical trial; and/or selection of a treatment for a subject with a disease or disorder.
Spatial information can provide information of biological importance. For example, the methods and compositions described herein can allow for: identification of transcriptome and/or proteome expression profiles (e.g., in healthy and/or diseased tissue); identification of multiple analyte types in close proximity (e.g., nearest neighbor analysis); determination of up- and/or 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 tissue; and identification of genetic variants within tissues (e.g., based on gene and/or protein expression profiles associated with specific disease or disorder biomarkers).
Typically, for spatial array-based methods, a substrate functions as a support for direct or indirect attachment of capture probes to features of the array. A “feature” is an entity that acts as a support or repository for various molecular entities used in spatial analysis. In some embodiments, some or all of the features in an array are functionalized for analyte capture. Exemplary substrates are described in Section (II)(c) of WO 2020/176788 and/or U.S. Patent Application Publication No. 2020/0277663. Exemplary features and geometric attributes of an array can be found in Sections (II)(d)(i), (II)(d)(iii), and (II)(d)(iv) of WO 2020/176788 and/or U.S. Patent Application Publication No. 2020/0277663.
Generally, analytes and/or intermediate agents (or portions thereof) can be captured when contacting a biological sample with a substrate including capture probes (e.g., a substrate with capture probes embedded, spotted, printed, fabricated on the substrate, or a substrate with features (e.g., beads, wells) comprising capture probes). As used herein, “contact,” “contacted,” and/or “contacting,” a biological sample with a substrate refers to any contact (e.g., direct or indirect) such that capture probes can interact (e.g., bind covalently or non-covalently (e.g., hybridize)) with analytes from the biological sample. Capture can be achieved actively (e.g., using electrophoresis) or passively (e.g., using diffusion).
In some cases, spatial analysis can be performed by attaching and/or introducing a molecule (e.g., a peptide, a lipid, or a nucleic acid molecule) having a barcode (e.g., a spatial barcode) to a biological sample (e.g., to a cell in a biological sample). In some embodiments, a plurality of molecules (e.g., a plurality of nucleic acid molecules) having a plurality of barcodes (e.g., a plurality of spatial barcodes) are introduced to a biological sample (e.g., to a plurality of cells in a biological sample) for use in spatial analysis. In some embodiments, after attaching and/or introducing a molecule having a barcode to a biological sample, the biological sample can be physically separated (e.g., dissociated) into single cells or cell groups for analysis. Some such methods of spatial analysis are described in Section (III) of WO 2020/176788 and/or U.S. Patent Application Publication No. 2020/0277663.
In some cases, spatial analysis can be performed by detecting multiple oligonucleotides that hybridize to an analyte. In some instances, for example, spatial analysis can be performed using RNA-templated ligation (RTL). Methods of RTL have been described previously. See, e.g., Credle et al., Nucleic Acids Res. 2017 Aug. 21; 45(14):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 domain (e.g., a poly(A) sequence, a non-homopolymeric sequence). After hybridization to the analyte, a ligase (e.g., SplintR ligase) ligates the two oligonucleotides together, creating a ligation product. In some instances, the two oligonucleotides hybridize to sequences that are not adjacent to one another. For example, hybridization of the two oligonucleotides creates a gap between the hybridized oligonucleotides. In some instances, a polymerase (e.g., a DNA polymerase) can extend one of the oligonucleotides prior to ligation. After ligation, the ligation product is released from the analyte. In some instances, the ligation product is released using an endonuclease (e.g., RNAse H). The released ligation product can then be captured by capture probes (e.g., instead of direct capture of an analyte) on an array, optionally amplified, and sequenced, thus determining the location and optionally the abundance of the analyte in the biological sample.
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 WO 2020/176788 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 WO 2020/176788 and/or U.S. Patent Application Publication No. 2020/0277663. See also, e.g., the Visium Spatial Gene Expression Reagent Kits User Guide (e.g., Rev C, dated June 2020), and/or the Visium Spatial Tissue Optimization Reagent Kits User Guide (e.g., Rev C, dated July 2020).
In some embodiments, spatial analysis can be performed using dedicated hardware and/or software, such as any of the systems described in Sections (II)(e)(ii) and/or (V) of WO 2020/176788 and/or U.S. Patent Application Publication No. 2020/0277663, or any of one or more of the devices or methods described in Sections Control Slide for Imaging, Methods of Using Control Slides and Substrates for, Systems of Using Control Slides and Substrates for Imaging, and/or Sample and Array Alignment Devices and Methods, Informational labels of WO 2020/123320.
Suitable systems for performing spatial analysis can include components such as a chamber (e.g., a flow cell or sealable, fluid-tight chamber) for containing a biological sample. The biological sample can be mounted for example, in a biological sample holder. One or more fluid chambers can be connected to the chamber and/or the sample holder via fluid conduits, and fluids can be delivered into the chamber and/or sample holder via fluidic pumps, vacuum sources, or other devices coupled to the fluid conduits that create a pressure gradient to drive fluid flow. One or more valves can also be connected to fluid conduits to regulate the flow of reagents from reservoirs to the chamber and/or sample holder.
The systems can optionally include a control unit that includes one or more electronic processors, an input interface, an output interface (such as a display), and a storage unit (e.g., a solid state storage medium such as, but not limited to, a magnetic, optical, or other solid state, persistent, writeable and/or re-writeable storage medium). The control unit can optionally be connected to one or more remote devices via a network. The control unit (and components thereof) can generally perform any of the steps and functions described herein. Where the system is connected to a remote device, the remote device (or devices) can perform any of the steps or features described herein. The systems can optionally include one or more detectors (e.g., CCD, CMOS) used to capture images. The systems can also optionally include one or more light sources (e.g., LED-based, diode-based, lasers) for illuminating a sample, a substrate with features, analytes from a biological sample captured on a substrate, and various control and calibration media.
The systems can optionally include software instructions encoded and/or implemented in one or more of tangible storage media and hardware components such as application specific integrated circuits. The software instructions, when executed by a control unit (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 Application No. 2020/061064 and/or U.S. patent application Ser. No. 16/951,854.
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 Application No. 2020/053655 and spatial analysis methods are generally described in WO 2020/061108 and/or U.S. patent application Ser. No. 16/951,864.
In some cases, a map of analyte presence and/or level can be aligned to an image of a biological sample using one or more fiducial markers, e.g., objects placed in the field of view of an imaging system which appear in the image produced, as described in the Substrate Attributes Section, Control Slide for Imaging Section of WO 2020/123320, PCT Application No. 2020/061066, and/or U.S. patent application Ser. No. 16/951,843. 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 distance.
II. Using Barcoded 5′ FLAP Probes for the Detection of gDNA
(a) DNA-Templated Ligation Using a 5′ FLAP Sequence
Templated ligation, or RNA templated ligation (RTL), is a process that includes multiple RTL probes (e.g., usually two oligonucleotides or probes) that hybridize to adjacent complementary mRNA sequences. RNA-templated ligation is enabled by the ability of RNases to remove the RNA in a RNA-DNA hybrid. Provided herein are methods that co-opt RTL principles to include oligonucleotide probes that hybridize to adjacent complementary genomic DNA (gDNA) sequences. It is known that DNA-DNA hybrids are harder to separate than RNA-DNA hybrids. Therefore, a readout of the DNA-templated ligation event is needed that can be measured without having to make single stranded DNA from the DNA-DNA hybrid. Here, an oligonucleotide (e.g., a second RTL probe) includes a 5′ FLAP sequence that can be removed and detected following an enzyme-mediated cleavage event that does not require removal of the hybridized probes. Detection of the 5′ FLAP provides a readout for the DNA-templated ligation event, which can serve as a proxy for detecting a gDNA analyte or a genetic variant within a gDNA analyte.
The methods provided herein rely on the cleavage of the 5′ FLAP from the second RTL probe occurring when the first RTL probe and the second RTL probe hybridize to a gDNA analyte at adjacent sequences. In some cases, the adjacent sequences flank a target sequence (e.g., a genetic variant). In some cases, the adjacent sequences directly abut each other in the gDNA analyte. Once a first RTL probe and a second RTL probe hybridize to a gDNA analyte, an enzyme-mediated cleavage event cleaves the second RTL probe, which includes non-complementary nucleotides (e.g., a 5′ FLAP). Upon cleavage of the 5′ FLAP, the sequence of the 5′ FLAP can be determined and used to detect the presence of a gDNA analyte in a biological sample or the presence of a genetic variant in a gDNA analyte. In some instances, determining the sequence of the 5′ FLAP includes hybridizing the 5′ FLAP to a probe on an array. In such cases, the 5′ FLAP includes a capture probe binding domain sequence (e.g., a poly-adenylation sequence or a target nucleic acid sequence) that can be detected by a capture probe on an array described herein (e.g., the capture probe comprises a poly-thymine sequence in some instances). In some instances, determining the sequence of the 5′ FLAP includes sequencing the 5′ FLAP without first hybridizing the 5′ FLAP to a capture probe on an array.
Thus, the methods provided herein allow detection of a gDNA analyte or a genetic variant in a gDNA analyte and in some cases enable detection of the spatial location within a biological sample. In addition, the methods provided herein allow detection of a gDNA analyte within a subset of DNA analytes, thereby increasing the resolution of the technology as applied to gDNA. Also, the methods provided herein avoid the need for probe ligation and endoribonuclease (e.g., RNaseH) treatment, which are both used in a standard RTL workflow, thereby keeping costs low without sacrificing efficiency.
Disclosed herein are methods for detecting an gDNA analyte in a biological sample where (i) the first RTL probe and second RTL probe hybridize to adjacent sequences on the gDNA analyte, (ii) enzyme-mediated cleavage of a 5′ FLAP results in release of the 5′ FLAP, and (iii) the sequence of the 5′ FLAP is determined and used to detect the gDNA in the biological sample. In some embodiments, the method includes detecting a gDNA analyte at a spatial location in a biological sample using a substrate to capture the released 5′ FLAP. In such cases, the method includes contacting the biological sample with a substrate that has a plurality of capture probes, where a capture probe of the plurality of capture probes includes a spatial barcode and a capture domain, prior to the RTL probe hybridization step. After cleavage of the 5′ FLAP, it hybridizes to the capture domain, and the sequence of the 5′ FLAP is determined. The along with determination of the spatial barcode, the spatial location of the gDNA analyte in the biological sample can be detected.
The methods disclosed herein use compositions comprising an RTL probe (e.g., a second RTL probe) that has a non-hybridizing sequence that can be used to carry information about the hybridization event between an analyte and an oligonucleotide. In some instances, the non-hybridizing sequence is referred to as a 5′ FLAP. See e.g.,
In some embodiments, the subset of analytes includes gDNA analytes including DNA that is transcribed into RNA (e.g., coding regions including, without limitation, gene exons, gene introns, and untranslated regions (both 5′ and 3′)) and DNA that is not transcribed into RNA but regulates transcription of DNA into RNA (e.g., regulatory sequences including, without limitation, promoters, enhancers, and silencers). Additional gDNA analytes include gDNA sequences associated with a particular region of a chromosome (e.g., without limitation, a telomere, a centromere, a topologically associated domain, an arm of a chromosome, a portion of an arm of a chromosome, a condensed chromosome, and an uncondensed chromosome).
In some embodiments, the subset of analytes includes 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, about 55, about 60, about 65, about 70, about 75, about 80, about 85, about 90, about 95, about 100, about 110, about 120, about 130, about 140, about 150, about 160, about 170, about 180, about 190, about 200, about 225, about 250, about 275, about 300, about 325, about 350, about 375, about 400, about 425, about 450, about 475, about 500, about 600, about 700, about 800, about 900, about 1000, or more analytes.
Methods disclosed herein can be performed on any type of sample. In some embodiments, the sample is a fresh tissue. In some embodiments, the sample is a frozen sample. In some embodiments, the sample was previously frozen. In some embodiments, the sample is a formalin-fixed, paraffin embedded (FFPE) sample. FFPE samples generally are heavily cross-linked and fragmented, and therefore this type of sample allows for limited DNA recovery using conventional detection techniques. In certain embodiments, methods of targeted DNA capture provided herein are less affected by DNA degradation associated with FFPE fixation than other methods (e.g., methods that take advantage of oligo-dT capture and reverse transcription of gDNA). In certain embodiments, methods provided herein enable sensitive measurement of specific genes of interest that otherwise might be missed with a whole transcriptomic approach.
In some embodiments, a biological sample (e.g. tissue section) can be fixed with methanol, stained with hematoxylin and eosin, and imaged. In some embodiments, fixing, staining, and imaging occurs before one or more RTL probes are hybridized to the sample. Some embodiments of any of the workflows described herein can further include a destaining step (e.g., a hematoxylin and eosin destaining step), after imaging of the sample and prior to permeabilizing the sample. For example, destaining can be performed by performing one or more (e.g., one, two, three, four, or five) washing steps (e.g., one or more (e.g., one, two, three, four, or five) washing steps performed using a buffer including HCl). The images can be used to map spatial gene expression patterns back to the biological sample. A permeabilization enzyme can be used to permeabilize the biological sample directly on the slide.
In some embodiments, the methods of targeted DNA capture as disclosed herein include hybridization of multiple RTL probes. In some embodiments, the methods include 2, 3, 4, or more RTL probes that hybridize to one or more analytes of interest. In some embodiments, the methods include two RTL probes. In some embodiments, the RTL probe includes sequences complementary that are complementary or substantially complementary to an analyte. For example, in some embodiments, the RTL probe includes a sequence that is complementary or substantially complementary to an analyte (e.g., a gDNA of interest (e.g., to a portion of the sequence of a gDNA of interest)). Methods provided herein may be applied to a single nucleic acid molecule or a plurality of nucleic acid molecules. A method of analyzing a sample comprising a nucleic acid molecule may comprise providing a plurality of nucleic acid molecules (e.g., DNA molecules), where each nucleic acid molecule comprises a first target region (e.g., a sequence that is 3′ of a target sequence or a sequence that is 5′ of a target sequence) and a second target region (e.g., a sequence that is 5′ of a target sequence or a sequence that is 3′ of a target sequence), a plurality of first RTL probes, and a plurality of second RTL probes.
After hybridization of the first and second RTL probes, the first RTL probe can be extended. After hybridization, in some embodiments, the second RTL probe can be extended. Extending probes can be accomplished using any method disclosed herein. In some instances, a polymerase (e.g., a DNA polymerase) extends the first and/or second RTL probe.
In some embodiments, methods disclosed herein include a wash step. In some instances, the wash step occurs after hybridizing the first and the second RTL probes. The wash step removes any unbound probes and can be performed using any technique or solution disclosed herein or known in the art. In some embodiments, multiple wash steps are performed to remove unbound RTL probes.
In some embodiments, after hybridization of RTL probes (e.g., first and the second RTL probes) to the gDNA analyte, the 5′ FLAP is cleaved, releasing the 5′ FLAP. Releasing the 5′ FLAP can be performed enzymatically as described herein. As disclosed in the following sections, releasing the 5′ FLAP allows for detection of a gDNA analyte of interest.
(i) gDNA Detection in a Biological Sample Using a 5′ FLAP
This disclosure features a method for detecting a gDNA analyte in a biological sample using methods that co-opt RTL principles to include RTL probes that hybridize to adjacent complementary genomic DNA (gDNA) sequences. In particular, the methods disclosed herein utilize a process that cleaves a non-hybridized sequence of one of the RTL probes. The non-hybridized sequence (e.g., a 5′ FLAP) can be further analyzed to determine the presence or absence of a gDNA analyte of interest.
A non-limiting example of a method for detecting a gDNA analyte in a biological sample includes: (a) contacting the biological sample with a first RTL probe and a second RTL probe, wherein the first RTL probe and the second RTL probe are substantially complementary to adjacent sequences of the gDNA analyte, and wherein the second RTL probe includes a 5′ FLAP; (b) hybridizing the first RTL probe and the second RTL probe to the gDNA analyte; (c) cleaving the second RTL probe, thereby releasing the 5′ FLAP; (d) determining all or a part of the sequence of the 5′ FLAP, and using the sequence of the 5′ FLAP to detect the gDNA analyte. In some embodiments, the method also includes denaturing the gDNA under conditions wherein the first RTL probe and the second RTL probe can hybridize to the gDNA analyte. In some embodiments, the method includes a denaturing step prior to contacting the biological sample with a first RTL probe and a second RTL probe. In some embodiments, the method further includes, prior to (a), contacting the biological sample with a substrate comprising capture probes. In some embodiments, the method further includes contacting the biological sample with a permeabilization reagent. In some embodiments, the biological sample is contacted with the permeabilization reagent before determining all or a part of the sequence of the 5′ FLAP, and using the sequence of the 5′ FLAP to detect the gDNA analyte. In some embodiments, the method also includes determining step includes amplifying all or part of the 5′ FLAP. In some embodiments, the amplifying includes isothermal amplification. In some embodiments, the determining step includes sequencing.
In some embodiments, the cleaving step includes contacting with an endonuclease. The endonuclease can include a 5′ FLAP endonuclease activity where the enzyme catalyzes the cleavage of a 5′ FLAP structure. The step uses two RTL probes that are hybridized to target nucleic acid (e.g., gDNA) containing, for example, a polymorphic site. One RTL probe is complementary to the target sequence 3′ of the polymorphic site and ends with a non-matching base overlapping the SNP nucleotide. The second RTL probe, the allele-specific probe, contains the complementary base of the SNP allele and extends to the sequence 5′ of the polymorphic site. This probe can also extend on its 5′ site with additional non-complementary nucleotides. An invasive cleavage structure is formed when the first RTL probe and the second RTL probe are both hybridized to the gDNA molecule. Once the two oligonucleotides anneal to the target DNA, they form a three-dimensional invasive structure over the SNP site that can be recognized by cleavase, a FEN enzyme. The enzyme cleaves the probe 3′ of the base complementary to the polymorphic site (i.e. 3′ of the overlapping invader structure). After hybridization, the invasive cleavage structure is formed when the first RTL probe and the second RTL probe are both hybridized to the gDNA analyte. Additional exemplary support for the invasive cleavage structure utilized in an Invader assay is provided in Olivier, Mutat. Res., 2005 Jun. 3; 573(1-2): 103-110, which is incorporated by reference in its entirety.
In some embodiments, the endonuclease cleaves a portion of the second RTL probe. In some embodiments, the endonuclease cleaves the 5′ FLAP of the second RTL probe. Non-limiting examples of endonucleases with 5′-FLAP endonuclease activity that can be used in the cleaving step include DNA replication helicase 2 (DNA2), Exonuclease 1 (EXO1), FANCD2/FANCI-associated nuclease 1 (FAN1), Holliday junction 5′ flap endonuclease (GEN1), structure-specific endonuclease subunit homolog B (SLX1B), and GRF-type containing 1 (ZGRF1). In some embodiments, the endonuclease is FLAP endonuclease 1 (FEN1).
In some embodiments, the cleaving step includes providing a DNA-dependent DNA polymerase. The DNA-dependent DNA polymerase can include 3′ to 5′ exonuclease activity. The DNA polymerase can include strand displacement properties. For example, the DNA polymerase uses the strand displacement property to displace the second RTL probe and an endonuclease cleaves the portion of the second RTL probe not bound (e.g., the 5′ FLAP and the portion of the second RTL probe displaced by the DNA polymerase) to the target sequence or the sequence 5′ of the target sequence. The portion of the second RTL probe not bound to the target sequence or the sequence 5′ of the target sequence includes the 5′ FLAP. Non-limiting examples of a DNA polymerase that can be used in the cleaving step include Phi29 DNA polymerase and Taq DNA polymerase.
In some embodiments, the 5′ FLAP further includes a first barcode sequence. In some embodiments, the first barcode sequence includes a sequence that identifies the first RTL probe, the second RTL probe, and/or the gDNA analyte. In some instances, the first barcode sequence is a sequence unique to the interaction between the first RTL probe, the second RTL probe, and/or the gDNA analyte. Thus, it can be decoded to determine the presence of the interaction between the first RTL probe, the second RTL probe, and/or the gDNA analyte. In some instances, the first barcode sequence can be used to identify the second RTL probe. When the 5′ FLAP is cleaved, the 5′ FLAP can be used to identify a hybridization event where both the first RTL probe and the second RTL probe hybridize to a gDNA analyte.
In some embodiments, the 5′ FLAP also includes a functional sequence. The functional sequence can be a primer sequence. The primer sequence can be used to amplify the 5′ FLAP before cleavage, contemporaneously with cleavage, or after cleavage of the 5′ FLAP from the second RTL probe.
In some embodiments, the second RTL probe includes a second barcode. For example, the second barcode is on the portion of the second RTL probe that is not released following cleavage. In some embodiments, the second barcode can be used to identify the second RTL probe.
This disclosure features a method for detecting a gDNA analyte at a spatial location in a biological sample using methods that co-opt RTL principles to include RTL probes that hybridize to adjacent complementary genomic DNA (gDNA) sequences. In particular, the methods disclosed herein utilizes a process that cleaves a non-hybridized sequence of an RTL probe. The non-hybridized sequence (e.g., a 5′ FLAP) can be further analyzed to determine the presence or absence of a gDNA analyte of interest.
In some instances, a method for detecting a gDNA analyte at a spatial location in a biological sample includes: (a) contacting the biological sample with a substrate including a plurality of capture probes, wherein a capture probe of the plurality includes a spatial barcode and a capture domain; (b) contacting a biological sample with a first RTL probe and a second RTL probe, wherein the first RTL probe and the second RTL probe are substantially complementary to adjacent sequences of the gDNA analyte, and wherein the second RTL probe includes a 5′ FLAP; (c) hybridizing the first RTL probe and the second RTL probe to the gDNA analyte; (d) cleaving the second RTL probe, thereby releasing the 5′ FLAP; (e) hybridizing the 5′ FLAP to the capture domain; and (f) determining (i) all or a part of the sequence of the 5′ FLAP, or a complement thereof, and (ii) all or a part of the sequence of the spatial barcode, or a complement thereof, and using the determined sequence of (i) and (ii) to identify the location of the gDNA analyte in the biological sample. In some embodiments, the method also includes denaturing the gDNA under conditions wherein the first RTL probe and the second RTL probe can hybridize to the gDNA analyte. In some embodiments, the method includes a denaturing step prior to contacting a biological sample with a first RTL probe and a second RTL probe. In some embodiments, the method further includes contacting the biological sample with a permeabilization reagent. In some embodiments, the biological sample is contacted with the permeabilization reagent before contacting the biological sample with a substrate including a plurality of capture probes. In some embodiments where the method includes detecting an analyte at a spatial location in a biological sample, the determining step includes amplifying all or part of the 5′ FLAP specifically bound to the capture domain. For example, the amplification can be isothermal. The determining step can include sequencing the 5′ FLAP.
In some embodiments, the cleaving step includes providing an endonuclease. The endonuclease can include a 5′ FLAP endonuclease activity where the enzyme catalyzes the cleavage of a 5′ FLAP structure. In some embodiments, the endonuclease cleaves a portion of second RTL probe. In some embodiments, the endonuclease cleaves the 5′ FLAP of the second RTL probe. Non-limiting examples of endonucleases with 5′-FLAP endonuclease activity that can be used in the cleaving step include DNA replication helicase 2 (DNA2), Exonuclease 1 (EXO1), FANCD2/FANCI-associated nuclease 1 (FAN1), Holliday junction 5′ flap endonuclease (GEN1), structure-specific endonuclease subunit homolog B (SLX1B), and GRF-type containing 1 (ZGRF1). In some embodiments, the endonuclease is FLAP endonuclease 1 (FEN1).
In some embodiments, the cleaving step includes providing a DNA-dependent DNA polymerase. The DNA-dependent DNA polymerase can include 3′ to 5′ exonuclease activity. The DNA polymerase can include strand displacement properties. For example, the DNA polymerase uses the strand displacement property to displace the second RTL probe and an endonuclease cleaves the portion of the second RTL probe not bound to the target sequence (e.g., the 5′ FLAP and the portion of the second RTL probe displaced by the DNA polymerase) or the sequence 5′ of the target sequence. The portion of the second RTL probe not bound to the target sequence or the sequence 5′ of the target sequence includes the 5′ FLAP. Non-limiting examples of DNA polymerase that can be used in the cleaving step include Phi29 DNA polymerase and Taq DNA polymerase.
In some embodiments, the method also includes providing a capture probe binding domain blocking moiety that interacts with the capture probe binding domain. In some embodiments, the method further includes releasing the capture probe binding domain blocking moiety from the capture probe binding domain prior to contacting the biological sample with the substrate. In some embodiments, the capture probe binding domain blocking moiety includes a poly-uridine sequence, a poly-thymidine sequence, or both. In some embodiments, releasing the capture probe binding domain blocking moiety from the poly(A) sequence includes denaturing the 5′ FLAP.
In some embodiments, the method for detecting an analyte at a spatial location in a biological sample includes a second RTL probe having a 5′ FLAP that further includes a first barcode sequence. In some embodiments, the first barcode sequence includes a sequence that identifies the first RTL probe, the second RTL probe, and/or the gDNA analyte. In some embodiments, the 5′FLAP further includes a functional sequence. For example, the functional sequence is a primer sequence.
In some embodiments, the 5′ FLAP further includes a capture probe binding domain. The capture domain includes a sequence that is substantially complementary to a capture domain on a capture probe where the capture probe binding domain sequence enables binding of the 5′ FLAP to the capture probe. The capture probe binding domain can include a homopolymeric sequence. For example, without limitation, the capture probe binding domain can be a poly(A) sequence.
In some embodiments, the method for detecting an analyte at a spatial location in a biological sample includes a second RTL probe that includes from 5′ to 3′: a 5′ FLAP and a sequence that is substantially complementary to a sequence that is adjacent to a sequence that is substantially complementary to a first RTL probe. In other cases, the second RTL probe includes a 5′ FLAP including from 5′ to 3′: a functional sequence, a first barcode, and a capture probe binding domain sequence.
In some embodiments, the second RTL probe further includes a second barcode. In some embodiments, the second barcode sequence includes a sequence that identifies the first RTL probe, the second RTL probe, and/or the gDNA analyte. In some embodiments, the second barcode sequence includes a sequence that identifies the second RTL probe. A second RTL probe can include from 5′ to 3′: a 5′ FLAP, a sequence that is substantially complementary to a sequence that is adjacent to a sequence that is substantially complementary to a first RTL probe, and a second barcode.
In some embodiments, the second RTL probe further includes a second capture probe binding domain. For example, a second RTL probe can include form 5′ to 3′: a 5′ FLAP, a sequence that is substantially complementary to a sequence that is adjacent to a sequence that is substantially complementary to a first RTL probe, a second barcode, and a second capture probe binding domain. The second capture domain can include any of the exemplary capture probe binding domain sequences describe herein. The second capture probe binding domain enables the portion of the second RTL probe that is not cleaved to hybridize to a capture domain on a capture probe.
(b) SNP Detection Using Templated Ligation and Detection of the 5′ FLAP
This disclosure also features methods of detecting a SNP using two oligonucleotide probes that are hybridized to a gDNA analyte containing a polymorphic site. The two oligonucleotides hybridize to the single-stranded target and form an overlapping invader structure at the site of the SNP. One oligonucleotide (e.g., a first RTL probe) is complementary to the target sequence 3′ of the polymorphic site and ends with a non-matching base overlapping the SNP nucleotide. The second oligonucleotide (e.g., the second RTL probe), the allele-specific probe, contains the complementary base of the SNP allele and extends to the sequence 5′ of the polymorphic site. In some instances, this second oligonucleotide can also include a 5′ FLAP including additional non-complementary nucleotides (e.g., a functional sequence and a barcode sequence). Once the two oligonucleotides hybridize to the target DNA, they form a three-dimensional invader structure over the SNP site that can be recognized by a cleavase, such as a FEN enzyme. An invasive cleavage structure is described in U.S. Pat. No. 6,913,881 B1, which is incorporated by reference in its entirety. The enzyme cleaves the 5′ FLAP (e.g., the non-complementary nucleotides) of the second RTL probe. Once cleaved, 5′ FLAP is released. Additional exemplary support for the invasive cleavage structure utilized in an Invader assay is provided in Olivier, Mutat. Res. 2005 Jun. 3; 573(1-2): 103-110, which is incorporated by reference in its entirety. In an alternative embodiment, the cleaved 5′ FLAP can be detected using a FRET pair, wherein the release of the FLAP results in fluorescence detection. In some instances, the 5′ FLAP includes a tag (e.g., a fluorescent tag) that can be detected upon cleavage. In another embodiment, after cleavage, the supernatant can be extracted and sequencing, providing the ability to detect the presence of a SNP or mutation of interest.
In some embodiments, a method for detecting a gDNA analyte in a biological sample includes (i) hybridizing a first RTL probe and a second RTL probe to adjacent sequences on the gDNA analyte, (ii) cleaving a 5′ FLAP from the second RTL probe, thereby releasing the 5′ FLAP, and (iii) determining the sequence of the 5′ FLAP and using the sequence of the 5′ FLAP to detect a genetic variant in the gDNA in the biological sample. In some embodiments, the method includes detecting a gDNA analyte at a spatial location in a biological sample using a substrate to capture the released 5′ FLAP. In such cases, the method further includes contacting the biological sample with a substrate including a plurality of capture probes, wherein, a capture probe of the plurality includes a spatial barcode and a capture domain, hybridizing the 5′ FLAP to the capture domain, and determining the sequence of the 5′ FLAP and using the sequence of the 5′ FLAP to detect a genetic variant in a gDNA analyte at a spatial location in the biological sample.
In some embodiments, a method of detecting a genetic variant in a gDNA analyte in a biological sample includes determining all or a part of the sequence of the 5′ FLAP, and using the sequence of the 5′ FLAP to detect the genetic variant in the gDNA analyte without using a substrate including capture probes to capture the 5′ FLAP. In such cases, the 5′ FLAP does not include a capture probe binding domain sequence. In such cases, a gDNA analyte is contacted with a first RTL probe and a second RTL probe, wherein the first RTL probe and the second RTL probe each include a sequence that is substantially complementary to adjacent sequences of the gDNA analyte; wherein the first RTL probe and the second RTL probe are capable of forming an invasive cleavage structure in the presence of the genetic variant; and wherein the second RTL probe further includes a 5′ FLAP. The 5′ FLAP can include a barcode sequence that can be used to identify the first RTL probe, the second RTL probe, and/or the gDNA analyte. In some cases, the method also includes denaturing the gDNA under conditions wherein the first RTL probe and the second RTL probe can hybridize to the gDNA analyte. The first RTL probe and the second RTL probe can hybridize to the gDNA analyte. Following hybridization in the presence of the genetic variant, the second RTL probe can be cleaved, thereby releasing the 5′ FLAP. Finally, all or a part of the sequence of the 5′ FLAP is determined, and used to detect the genetic variant in the gDNA analyte.
In another embodiment, this disclosure features a method of detecting a genetic variant in a gDNA analyte at a spatial location in a biological sample. In a non-limiting example this method includes contacting the biological sample with a first RTL probe and a second RTL probe, wherein the first RTL probe and the second RTL probe each include a sequence that is substantially complementary to adjacent sequences of the gDNA analyte; wherein the first RTL probe and the second RTL probe are capable of forming an invasive cleavage structure when the genetic variant is present; and wherein the second RTL probe further includes a 5′ FLAP. The 5′ FLAP can include a barcode sequence that can be used to identify the first RTL probe, the second RTL probe, and/or the gDNA analyte and a capture probe binding domain sequence that can be used to hybridize the 5′ FLAP to capture domain of a capture probe located on the surface of a substrate. In some cases, the method also includes denaturing the gDNA under conditions wherein the first RTL probe and the second RTL probe can hybridize to the gDNA analyte. Following hybridization of the first RTL probe and the second RTL probe in the presence of the genetic variant, the second RTL probe can be cleaved, thereby releasing the 5′ FLAP. The method then includes contacting the biological sample with a substrate including a plurality of capture probes, wherein a capture probe of the plurality includes a spatial barcode and a capture domain; hybridizing the 5′ FLAP to the capture domain; and determining (i) all or a part of the sequence of the 5′ FLAP, or a complement thereof, and (ii) all or a part of the sequence of the spatial barcode, or a complement thereof, and using the determined sequence of (i) and (ii) to identify the genetic variant in the gDNA analyte at a spatial location in the biological sample.
In some embodiments, the first RTL probe includes a sequence that is substantially complementary to a sequence 3′ of the target sequence (e.g., the genetic variant). In some embodiments, the first RTL probe is at least 70% identical (e.g., at least 75% identical, at least 80% identical, at least 85% identical, at least 90% identical, at least 95% identical, or at least 99% identical) to a sequence 3′ of the target sequence (e.g., the genetic variant).
In some embodiments, the first RTL probe includes a sequence that is substantially complementary to a sequence 5′ of the target sequence (e.g., the genetic variant). In some embodiments, the first RTL probe is at least 70% identical (e.g., at least 75% identical, at least 80% identical, at least 85% identical, at least 90% identical, at least 95% identical, or at least 99% identical) to a sequence 5′ of the target sequence (e.g., the genetic variant).
In some embodiments, the first RTL probe includes at least one nucleotide that is complementary to a reference target sequence (e.g., wild type sequence of a genetic variant). In some embodiments, the first RTL probe includes at least one nucleotide that is complementary to a single nucleotide polymorphism (e.g., a single nucleotide polymorphism as compared to a reference target sequence).
In some embodiments, the second RTL probe includes a sequence substantially complementary to a sequence 5′ to the target sequence (e.g., the genetic variant). In some embodiments, the second RTL probe is at least 70% identical (e.g., at least 75% identical, at least 80% identical, at least 85% identical, at least 90% identical, at least 95% identical, or at least 99% identical) to a sequence 5′ of the target sequence (e.g., the genetic variant).
In some embodiments, the second RTL probe includes a sequence substantially complementary to a sequence 3′ to the target sequence (e.g., the genetic variant). In some embodiments, the second RTL probe is at least 70% identical (e.g., at least 75% identical, at least 80% identical, at least 85% identical, at least 90% identical, at least 95% identical, or at least 99% identical) to a sequence 3′ of the target sequence (e.g., the genetic variant).
In some embodiments, the second RTL probe includes at least one nucleotide complementary to the at least one genetic variant of the target sequence. In some embodiments, the second RTL probe includes at least one nucleotide complementary to a reference target sequence (e.g., a wild type sequence).
In some embodiments, the second RTL probe includes from 5′ to 3′: a sequence of non-complementary nucleotides (e.g., a 5′ FLAP), one or more nucleotides complementary to the target sequence (e.g., the genetic variant), and a sequence substantially complementary to a sequence 5′ of the target sequence (e.g., the genetic variant). In such cases, the 5′ FLAP includes a barcode sequence. Where the 5′ FLAP sequence is determined without hybridizing the 5′ FLAP to a capture probe, the 5′ FLAP does not include a capture probe binding domain sequence. In some cases where the 5′ FLAP sequence is determined without hybridizing the 5′ FLAP to a capture probe, the 5′ FLAP includes a capture probe binding domain sequence. In cases where the sequence of the 5′ FLAP is determined by hybridizing the 5′ FLAP to a capture probe (i.e., via a capture domain), the 5′ FLAP includes a capture probe binding domain sequence.
In some embodiments, the target sequence in an analyte of interest includes one or more nucleotides. In some embodiments, the target sequence includes one or more single nucleotide variants (SNV) compared to a reference target sequence. In some instances, the target sequence includes one SNV compared to a reference target sequence. In some embodiments, the at least one genetic variant is a single nucleotide polymorphism (SNP). For example, a gDNA analyte can include one or more SNPs compared to a reference gDNA analyte. In some embodiments, the at least one genetic variant is a nucleotide point mutation. In some embodiments, the at least one genetic variant includes at least two, at least three, at least four, at least five, or more genetic variants.
In some embodiments, after formation of the invader structure, the biological sample is contacted with an endonuclease. In some instances, the endonuclease cleaves the sequence of non-complementary nucleotides (e.g. a 5′ FLAP) of the second RTL probe. In some embodiments, the endonuclease cleaves a portion of the first RTL probe. In some embodiments, the endonuclease is FLAP endonuclease 1 (FEN1). FEN1 is a structure-specific endonuclease that cuts at the base of single-stranded FLAPs. See Balakrishnan and Bambara, Annu Rev Biochem. 2013 Jun. 2; 82: 119-138, which is incorporated by reference in its entirety. In some embodiments, the endonuclease cleaves the at least one nucleotide that is complementary to a wild-type sequence of the target sequence of the first RTL probe.
In some instances, once FEN1 cleaves the 5′ FLAP, the hybridized RTL probes are ligated using methods disclosed herein (e.g., enzymatically using e.g., T4 DNA ligase or chemically). In some instances, the ligation step comprises ligating the first RTL probe and the 3′ end of a gap RTL probe using enzymatic or chemical ligation. In some instances, the ligation step comprises ligating the second RTL probe and the 5′ end of a gap RTL probe using enzymatic or chemical ligation. In some instances, the enzymatic ligation utilizes a ligase. In some instances, the ligase is one or more of a T4 DNA ligase (Rnl2), a SplintR ligase, a single stranded DNA ligase, or a T4 DNA ligase. In some instances, the ligase is a T4 DNA ligase 2 (Rnl2) ligase. In some instances, the ligase is a pre-activated T4 DNA ligase. In some embodiments, after releasing the 5′ FLAP from the second RTL probe, the biological sample is permeabilized. In some embodiments, permeabilization occurs using a protease (e.g., an endopeptidase disclosed herein).
SNP Detection in a Biological Sample Using a 5′ FLAP
This disclosure also features a method of detecting a genetic variant in gDNA analyte in a biological sample where the method includes determining all or a part of the sequence of the 5′ FLAP, and using the sequence of the 5′ FLAP to detect the genetic variant in the gDNA analyte without using a substrate including capture probes to capture the 5′ FLAP.
In another aspect, this disclosure features a method of detecting a genetic variant in a gDNA analyte in a biological sample including: (a) contacting the gDNA analyte with a first RTL probe and a second RTL probe, wherein the first RTL probe and the second RTL probe each include a sequence that is substantially complementary to adjacent sequences of the gDNA analyte; wherein the first RTL probe and the second RTL probe are capable of forming an invasive cleavage structure in the presence of the genetic variant; and wherein the second RTL probe further includes a 5′ FLAP; (b) hybridizing the first RTL probe and the second RTL probe to the gDNA analyte; (c) cleaving the second RTL probe when the genetic variant is present, thereby releasing the 5′ FLAP; and (d) determining all or a part of the sequence of the 5′ FLAP, and using the sequence of the 5′ FLAP to detect the genetic variant in the gDNA analyte. In some embodiments, the method also includes denaturing the gDNA under conditions wherein the first RTL probe and the second RTL probe can hybridize to the gDNA analyte. In some embodiments, the method includes a denaturing step prior to contacting the gDNA analyte with a first RTL probe and a second RTL probe. In some embodiments, the method further includes contacting the biological sample with a permeabilization reagent. In some embodiments, the biological sample is contacted with the permeabilization reagent determining all or a part of the sequence of the 5′ FLAP, and using the sequence of the 5′ FLAP to detect the genetic variant in the gDNA analyte. In some embodiments, the method also includes a determining step including amplifying all or part of the 5′ FLAP. In some embodiments, the amplifying includes isothermal amplification. In some embodiments, the determining step includes sequencing.
In some embodiments, the cleaving step includes providing an endonuclease. In some embodiments, the endonuclease cleaves the invasive cleavage structure. In some embodiments, the endonuclease cleaves a portion of the first RTL probe. In some embodiments, the endonuclease cleaves the at least one nucleotide that is complementary to a wild-type sequence of the gDNA analyte of the first RTL probe. In some embodiments, the endonuclease cleaves a portion of the second RTL probe. In some embodiments, the endonuclease cleaves the 5′ FLAP of the second RTL probe. In some embodiments, the endonuclease is FLAP endonuclease 1 (FEN1).
In some embodiments, the 5′ FLAP further includes a first barcode sequence. In some embodiments, the first barcode sequence includes a sequence that identifies the first RTL probe, the second RTL probe, and/or the gDNA analyte. When the 5′ FLAP is cleaved, the 5′ FLAP can be used to identify a hybridization event where both the first RTL probe and the second RTL probe hybridize to a gDNA analyte.
In some embodiments, the 5′ FLAP also includes a functional sequence. The functional sequence can be a primer sequence. The primer sequence can be used to amplify the 5′ FLAP before cleavage, contemporaneously with cleavage, or after cleavage of the 5′ FLAP from the second RTL probe. The functional sequence can be a sequence for use in sequencing the FLAP.
In some embodiments, when the method comprises cleaving the second RTL probe when the genetic variant is present, the 5′ FLAP includes a nucleotide that is complementary to the genetic variant. In some embodiments, following cleavage of the 5′ FLAP, the portion of the second RTL probe that is not released includes a nucleotide that is complementary to the genetic variant.
In some embodiments, the second RTL probe includes a second barcode. For example, the second barcode is on the portion of the second RTL probe that is not released following cleavage. In some embodiments, the second barcode can be used to identify the second RTL probe.
This disclosure features methods of detecting a genetic variant in a gDNA analyte at a spatial location in a biological sample using a substrate to capture the released 5′ FLAP. In such cases, the method includes contacting the biological sample with a substrate including a plurality of capture probes, where, a capture probe of the plurality includes a spatial barcode and a capture domain, hybridizing the 5′ FLAP to the capture domain, and determining the sequence of the 5′ FLAP and using the sequence of the 5′ FLAP to detect a genetic variant in a gDNA analyte at a spatial location in the biological sample.
A non-limiting example of a method of detecting a genetic variant in a gDNA analyte at a spatial location in a biological sample includes: (a) contacting the biological sample with a first RTL probe and a second RTL probe, wherein the first RTL probe and the second RTL probe each include a sequence that is substantially complementary to adjacent sequences of the gDNA analyte; wherein the first RTL probe and the second RTL probe are capable of forming an invasive cleavage structure when the genetic variant is present; and wherein the second RTL probe further includes a 5′ FLAP; (b) hybridizing the first RTL probe and the second RTL probe to the gDNA analyte; (c) cleaving the second RTL probe when the genetic variant is present, thereby releasing the 5′ FLAP; (d) contacting the biological sample with a substrate including a plurality of capture probes, wherein a capture probe of the plurality includes a spatial barcode and a capture domain; (e) hybridizing the 5′ FLAP to the capture domain; and (f) determining (i) all or a part of the sequence of the 5′ FLAP, or a complement thereof, and (ii) all or a part of the sequence of the spatial barcode, or a complement thereof, and using the determined sequence of (i) and (ii) to identify the spatial location of the gDNA analyte in the biological sample. In some embodiments, the method also includes denaturing the gDNA under conditions wherein the first RTL probe and the second RTL probe can hybridize to the gDNA analyte. In some embodiments, the method includes a denaturing step prior to contacting the biological sample with a first RTL probe and a second RTL probe. In some embodiments, the method further includes contacting the biological sample with a permeabilization reagent. In some embodiments, the biological sample is contacted with the permeabilization reagent before determining all or a part of the sequence of the 5′ FLAP, and using the sequence of the 5′ FLAP to detect the genetic variant in the gDNA analyte.
In some embodiments, the cleaving step includes contacting with an endonuclease. In some embodiments, the endonuclease cleaves the invasive cleavage structure. In some embodiments, the endonuclease cleaves a portion of the first RTL probe. In some embodiments, the endonuclease cleaves the at least one nucleotide that is complementary to a wild-type sequence of the gDNA analyte of the first RTL probe. In some embodiments, the endonuclease cleaves a portion of second RTL probe. In some embodiments, the endonuclease cleaves the 5′ FLAP of the second RTL probe. In some embodiments, the endonuclease is FLAP endonuclease 1 (FEN1).
In some embodiments, the method also includes providing a capture probe binding domain blocking moiety that interacts with the capture probe binding domain. In some embodiments, the method further includes releasing the capture probe binding domain blocking moiety from the capture probe binding domain prior to contacting the biological sample with the substrate. In some embodiments, the capture probe binding domain blocking moiety includes a poly-uridine sequence, a poly-thymidine sequence, or both. In some embodiments, releasing the capture probe binding domain blocking moiety from the poly(A) sequence includes denaturing the 5′ FLAP.
In some embodiments where the method includes detecting a genetic variant in a gDNA analyte at a spatial location in a biological sample, the determining step includes amplifying all or part of the 5′ FLAP specifically bound to the capture domain. For example, the amplification can be isothermal. In some embodiments, the determining step includes sequencing the 5′ FLAP.
In some embodiments, the method of detecting a genetic variant in a gDNA analyte at a spatial location in a biological sample includes a second RTL probe having a 5′ FLAP that further includes a first barcode sequence. In some embodiments, the first barcode sequence includes a sequence that identifies the first RTL probe, the second RTL probe, and/or the gDNA analyte. In some embodiments, the 5′FLAP further includes a functional sequence. For example, the functional sequence is a primer sequence. In some embodiments, the 5′ FLAP further includes a capture probe binding domain. The capture probe binding domain can include a homopolymeric sequence. For example, without limitation, the capture probe binding domain can be a poly(A) sequence.
In some embodiments, following cleavage of the 5′ FLAP, the 5′ FLAP includes a nucleotide that is complementary to the genetic variant. In some embodiments, following cleavage of the 5′ FLAP, the portion of the second RTL probe that is not released by the cleavage includes a nucleotide that is complementary to the genetic variant.
In some embodiments, the method of detecting a genetic variant in an gDNA analyte at a spatial location in a biological sample includes a second RTL probe that includes from 5′ to 3′: a 5′ FLAP and a sequence that is substantially complementary to a sequence that is adjacent to a sequence that is substantially complementary to a first RTL probe.
In some embodiments, the second RTL probe includes a 5′ FLAP including from 5′ to 3′: a functional sequence, a barcode, and a capture probe binding domain sequence. In some embodiments, the 5′ FLAP includes from 5′ to 3′: a functional sequence, a barcode, a capture probe binding domain sequence, and an additional nucleotide. In some embodiments, the additional nucleotide includes a nucleotide that is complementary to the genetic variant. In some embodiments, the additional nucleotide includes a nucleotide that is complementary to the wild type sequence of the genetic variant. In some embodiments, the second RTL probe further includes a nucleotide that is complementary to the genetic variant.
In some embodiments, the second RTL probe further includes a second barcode. In some embodiments, the second barcode sequence includes a sequence that identifies the first RTL probe, the second RTL probe, and/or the gDNA analyte. In some embodiments, the second barcode sequence includes a sequence that identifies the second RTL probe. A second RTL probe can include from 5′ to 3′: a 5′ FLAP, a sequence that is substantially complementary to a sequence that is adjacent to a sequence that is substantially complementary to a first RTL probe, and a second barcode. In some embodiments, the second RTL probe further includes a second capture probe binding domain. For example, a second RTL probe can include from 5′ to 3′: a 5′ FLAP, a sequence that is substantially complementary to a sequence that is adjacent to a sequence that is substantially complementary to a first RTL probe, a second barcode, and a second capture probe binding domain.
(c) Hybridization of 5′ FLAP to Capture Domain
In some embodiments, the 5′ FLAP includes a capture probe binding domain, which can hybridize to a capture probe (e.g., a capture probe immobilized, directly or indirectly, on a substrate). In some embodiments, methods provided herein include contacting a biological sample with a substrate, where the capture probe is affixed to the substrate (e.g., immobilized to the substrate, directly or indirectly). In some embodiments, the capture probe includes a spatial barcode and a capture domain. In some embodiments, the capture probe binding domain of the 5′ FLAP specifically binds to the capture domain. After hybridization of the 5′ FLAP to the capture probe, the 5′ FLAP 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 embodiments where the 5′ FLAP hybridizes to a capture probe on a substrate, the 5′ FLAP is extended to generate an extended 5′ FLAP. As shown in
In some embodiments where the 5′ FLAP includes an additional nucleotide at the 3′ end, extending the 5′ FLAP includes using a polymerase having a 3′ to 5′ exonuclease activity to remove the additional nucleotide sequence from the 3′ end and then extend the 5′ FLAP. The resulting extended 5′ FLAP 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 extended 5′ FLAP can be amplified via PCR prior to library construction. P5, P7, i7, and i5 can be incorporated into the library as for downstream sequencing, and additional library sequencing regions, such as TruSeq Read 2, can be added via End Repair, A-tailing, Adaptor Ligation, and PCR. The cDNA fragments can then be sequenced using paired-end sequencing using TruSeq Read 1 and TruSeq Read 2 as sequencing primer sites. In some instances, the cDNA library is sequenced using any method described herein, such that different sequencing domains specific to other sequencing methods and techniques can be incorporated into a capture probe or introduced during library preparation. In some instances, the sequence of the RTL product is determined via sequencing. In some instances, the spatial barcode is sequenced, providing the location of the gDNA analyte.
(d) RTL Probe Sets Comprising a 5′ FLAP on One Probe
Provided herein are templated ligation probes, or RTL probes, that hybridize to adjacent sequences of a target gDNA analyte. Compared to situations of templated ligation where the probes are ligated at each one's terminal end, herein, one of the probes includes a sequence that is not complementary to the target gDNA analyte, and thus overhangs on the side adjacent to the other RTL probes upon RTL probe hybridization.
In some instances, a set of RTL probes comprises a first RTL probe and a second RTL probe. In some instances, the first RTL probe comprises a primer sequence and a sequence complementary to a target analyte (e.g., gDNA). In some instances, the second RTL probe includes a 5′ FLAP, a sequence complementary to a target analyte (e.g., gDNA), and a poly(A) sequence. Detailed descriptions of RTL probes has been disclosed in WO 2021/133849, the entirety of which is incorporated herein by reference.
In some embodiments, one of the RTL probes (e.g., the second RTL probe) includes a sequence of non-complementary nucleotides (e.g. a 5′ FLAP). In some embodiments, non-complementary nucleotides include ten nucleotides, 11 nucleotides, 12 nucleotides, 13 nucleotides, 14 nucleotides, 15 nucleotides, 16 nucleotides, 17 nucleotides, 18 nucleotides, 19 nucleotides, 20 nucleotides, 21 nucleotides, 22 nucleotides, 23 nucleotides, 24 nucleotides, 25 nucleotides, 26 nucleotides, 27 nucleotides, 28 nucleotides, 29 nucleotides, 30 nucleotides, 31 nucleotides, 32 nucleotides, 33 nucleotides, 34 nucleotides, 35 nucleotides, 36 nucleotides, 37 nucleotides, 38 nucleotides, 39 nucleotides, 40 nucleotides, 41 nucleotides, 42 nucleotides, 43 nucleotides, 44 nucleotides, 45 nucleotides, 46 nucleotides, 47 nucleotides, 48 nucleotides, 49 nucleotides, 50 nucleotides, 51 nucleotides, 52 nucleotides, 53 nucleotides, 54 nucleotides, 55 nucleotides, 56 nucleotides, 57 nucleotides, 58 nucleotides, 59 nucleotides, or at least 60 nucleotides.
In some instances, the 5′ FLAP includes a functional sequence. The functional sequence can be a primer sequence. Other non-limiting examples of functional sequences includes Read 1 or Read 2 sequences (e.g., sequences that can be added for sequencing library preparation), index sequences such as an i5 sequence, and/or an i7 sequence (e.g., sequences complementary to P5 and P7), or any other sequence that facilitates detection of the 5′ FLAP. In some embodiments, the functional sequence can be a primer sequence where a primer hybridizes to the primer sequence and is used to amplify the 5′ FLAP. The 5′ FLAP can be amplified using any form of amplification, for example, linear amplification, PCR, of isothermal amplification, and the like.
In some instances, the 5′ FLAP includes a barcode sequence. The barcode sequence is a nucleic acid sequence that functions as a label or identifier of the 5′ FLAP. The barcode sequence can also function as a label of the first RTL probe, the second RTL probe, and/or the gDNA analyte to which the first RTL probe and the second RTL probe hybridize.
In some instances, the 5′ FLAP includes a capture probe binding domain sequence (e.g., any of the exemplary capture probe binding domain sequences described herein). The capture probe binding domain enables the 5′ FLAP to hybridize to a capture domain on a capture probe. In some cases, the capture probe is located on the surface of a substrate and includes a spatial barcode and a capture domain. In some embodiments, the capture probe binding domain includes a poly-uridine sequence, a poly-thymidine sequence, or both. In some embodiments, the capture probe binding domain includes a poly(A) sequence. In some embodiments, the capture probe binding domain includes a random sequence (e.g., a random hexamer or octamer). In some embodiments, the capture probe binding domain is complementary to a capture domain in a capture probe that detects a particular target(s) of interest. In some instances, the 5′ FLAP does not include a capture probe binding domain sequence. In this instance, identification of the 5′ FLAP is performed via downstream analysis (e.g., sequencing the 5′ FLAP or a complement thereof without hybridization to a probe on an array).
In some embodiments, the 5′ FLAP includes an additional nucleotide on its 3′ end. For example, the 5′ FLAP includes from 5′ to 3′: a functional sequence, a barcode, a capture probe binding domain sequence, and an additional nucleotide. The additional nucleotide can include a nucleotide that is complementary to the target sequence (e.g., the genetic variant). Alternatively, the additional nucleotide can include a nucleotide that is complementary to the target sequence (e.g., wild type sequence of the genetic variant). In some embodiments, the 5′ FLAP includes an additional two, there, four or five or more nucleotides on its 3′ end.
In some embodiments, the subset of analytes includes an individual target DNA. In some instances, the presence of the 5′ FLAP that is created as a result of the methods described herein indicates that the individual target DNA is present. In some instances, the absence of the 5′ FLAP that is created as a result of the RTL methods described herein indicates that the individual target DNA is present. In some instances, an absence of the 5′ FLAP is because one of the RTL probes did not hybridize to the gDNA analyte. In some instances, an absence of the 5′ FLAP is because both (e.g., two) of the RTL probes did not hybridize to the gDNA analyte.
In some embodiments, the subset of analytes detected using methods disclosed herein includes two or more (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10, or more) targeted DNAs. In some instances, the detection of analytes includes detection of one or more mutations in a gDNA analyte. In some embodiments, the subset of analytes includes detection of gDNAs having one or more single nucleotide polymorphisms (SNPs) in a biological sample.
In some embodiments, the methods that allow for targeted DNA capture as provided herein include a first RTL probe and a second RTL probe. The first and second RTL probes each include sequences that are substantially complementary to the sequence of an analyte of interest. By substantially complementary, it is meant that the first and/or second RTL probe is at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% complementary to a sequence in an DNA molecule. In some instances, the first RTL probe and the second RTL probe hybridize to adjacent sequences on a gDNA analyte. It is appreciated that the terms first RTL probe and the second RTL probe generally can be used interchangeably.
In some embodiments, the first and/or second probe as disclosed herein include a combination of ribonucleic acids and deoxyribonucleic acids. In some instances of the first and/or second RTL probes, the sequence that is substantially complementary to the sequence of the analyte of interest includes ribonucleic acids (e.g., does not include deoxyribonucleic acids). In some instances of the first and/or second RTL probes, the sequence that is substantially complementary to the sequence of the analyte of interest includes deoxyribonucleic acids (e.g., does not include ribonucleic acids).
In some embodiments, the first and/or second RTL probe as disclosed herein includes one of at least two ribonucleic acid bases at the 3′ end; a functional sequence; a phosphorylated nucleotide at the 5′ end; and/or a capture probe binding domain. In some embodiments, the first and/or second RTL probe as disclosed herein includes one of at least two deoxyribonucleic acid bases at the 3′ end; a functional sequence; a phosphorylated nucleotide at the 5′ end; and/or a capture probe binding domain. In some embodiments, the functional sequence is a primer sequence. The capture probe binding domain is a sequence that is complementary to a particular capture domain present in a capture probe. In some embodiments, the capture probe binding domain includes a poly(A) sequence. In some embodiments, the capture probe binding domain includes a poly-uridine sequence, a poly-thymidine sequence, or both. In some embodiments, the capture probe binding domain includes a random sequence (e.g., a random hexamer or octamer). In some embodiments, the capture probe binding domain is complementary to a capture domain in a capture probe that detects a particular target(s) of interest.
In some embodiments, a capture probe binding domain blocking moiety that interacts with the capture probe binding domain is provided. In some instances, the capture probe binding domain blocking moiety includes a nucleic acid sequence. In some instances, the capture probe binding domain blocking moiety is a DNA oligonucleotide. In some instances, the capture probe binding domain blocking moiety is an RNA oligonucleotide. In some embodiments, a capture probe binding domain blocking moiety includes a sequence that is complementary or substantially complementary to a capture probe binding domain. In some embodiments, a capture probe binding domain blocking moiety prevents the capture probe binding domain from binding the capture probe when present. In some embodiments, a capture probe binding domain blocking moiety is removed prior to binding the capture probe binding domain (e.g., present in a 5′ FLAP) to a capture probe. In some embodiments, a capture probe binding domain blocking moiety comprises a poly-uridine sequence, a poly-thymidine sequence, or both.
(e) Biological Samples
Methods disclosed herein can be performed on any type of biological sample, or sample. In some embodiments, the sample is a fresh tissue. In some embodiments, the sample is a frozen sample. In some embodiments, the sample was previously frozen. In some embodiments, the sample is a formalin-fixed, paraffin embedded (FFPE) sample. In certain embodiments, methods provided herein enable sensitive measurement of specific genes of interest that otherwise might be missed with a whole transcriptomic approach.
In some embodiments, a biological sample (e.g. tissue section) can be fixed with methanol, stained with hematoxylin and eosin, and imaged. In some embodiments, fixing, staining, and imaging occurs before one or more oligonucleotide probes are hybridized to the sample. Some embodiments of any of the workflows described herein can further include a destaining step (e.g., a hematoxylin and eosin destaining step), after imaging of the sample and prior to permeabilizing the sample. For example, destaining can be performed by performing one or more (e.g., one, two, three, four, or five) washing steps (e.g., one or more (e.g., one, two, three, four, or five) washing steps performed using a buffer including HCl). The images can be used to map spatial gene expression patterns back to the biological sample. A permeabilization enzyme can be used to permeabilize the biological sample directly on the slide.
(f) Compositions and Kits
In some instances, disclosed herein are compositions and systems that are used to carry out the methods described herein. In some instances, the kit includes a first RTL probe and a second RTL probe. In some instances, the first RTL probe and the second RTL probe in the kit are substantially complementary to adjacent sequences of an analyte. In some instances, the second RTL probe includes a 5′ FLAP that is not complementary to the analyte.
In some instances, the kit includes multiple sets of RTL probes, allowing for detection of multiple analytes using one kit. For example, in some instances, the kit includes at least 2 sets, at least 3 sets, at least 4 sets, at least 5 sets, at least 6 sets, at least 7 sets, at least 8 sets, at least 9 sets, or at least 10 sets, or more sets of RTL probes. It is appreciated that multiple sets of probes can detect nucleic acids or variants that are associated with particular physiologies or pathophysiologies (e.g., cancer).
In some instances, the kit includes one or more enzymes, including an endonuclease. In some instances, the endonuclease is FEN1. In some instances, the endonuclease cleaves the 5′ FLAP thereby releasing the 5′ FLAP from the second RTL probe.
In some instances, the kit further includes a substrate comprising a plurality of capture probes, wherein a capture probe of the plurality comprises a spatial barcode and a capture domain, wherein the 5′ FLAP is capable of hybridizing to the capture domain. It is appreciated that the kit can include any of the elements of the substrate, array, or capture probes as described herein.
In some instances, the kit further includes instructions for carrying out the methods of detecting a 5′ FLAP as described herein.
In some instances, compositions and systems are further provided herein. In some instances, a composition or system provided herein includes a first RTL probe and a second RTL probe hybridized to an analyte, wherein the first RTL probe and the second RTL probe are substantially complementary to adjacent sequences of the analyte, and wherein the second RTL probe comprises a 5′ FLAP. In some instances, a composition or system provided herein includes a first RTL probe and a second RTL probe hybridized to an analyte, wherein the first RTL probe and the second RTL probe each comprise a sequence that is substantially complementary to adjacent sequences of the gDNA analyte; wherein the first RTL probe and the second RTL probe are capable of forming an invasive cleavage structure in the presence of the genetic variant; and wherein the second RTL probe further comprises a 5′ FLAP. In some instances, the 5′ FLAP comprises a sequence that is capable of hybridizing to a capture domain of a capture probe. In some instances, the capture probe further comprises a spatial barcode. In some instances, the capture probe is part of a plurality of capture probes affixed to any one of the substrates or arrays as described throughout this disclosure.
This example provides an exemplary method of determining the presence or absence of an analyte in a biological sample. In a non-limiting example, a first RTL probe and a second RTL probe are substantially complementary to adjacent sequences of the gDNA analyte, and wherein the second RTL probe includes a 5′ FLAP. In the presence of the gDNA analyte in the biological sample, the first and second probes hybridize to the gDNA analyte, the second RTL probe is cleaved thereby releasing the 5′ FLAP. The sequence of the 5′ FLAP is determined and the determined sequence of the 5′ FLAP is used to detect the gDNA analyte.
As shown in
This example provides an exemplary method for determining the presence or absence of a genetic variant in a gDNA analyte in a biological sample. In a non-limiting example, a first RTL probe having a non-complementary nucleotide overlapping the target sequence (e.g., genetic variant) and a second RTL probe having a sequence complementary to the target sequence (e.g., genetic variant) are used to determine the presence or absence of a genetic variant in a biological sample. Others have demonstrated SNP detection using invader assays. Non-limiting aspects of SNP detection with invader assays are described in U.S. Pat. Nos. 7,011,944, 6,913,881, 6,875,572 and 6,872,816, each of which is incorporated by reference in its entirety and each of which can be used herein in any combination.
As shown in
This example provides an exemplary method for determining the location of an analyte in a biological sample using a first RTL probe and a second RTL probe.
In a non-limiting example, a first RTL probe and a second RTL probe are substantially complementary to adjacent sequences of the gDNA analyte, and wherein the second RTL probe includes a 5′ FLAP. In the presence of the gDNA analyte in the biological sample, the first and second probes hybridize to the gDNA analyte, the second RTL probe is cleaved thereby releasing the 5′ FLAP. The sequence of the 5′ FLAP is used to detect the gDNA analyte.
As shown in
As shown in
This example provides an exemplary method for determining the presence or absence of a genetic variant in a gDNA analyte in a biological sample. In a non-limiting example, a first RTL probe having a non-complementary nucleotide overlapping the target sequence (e.g., genetic variant) and a second RTL probe having a sequence complementary to the target sequence (e.g., genetic variant) are used to determine the location of a genetic variant in a biological sample.
As shown in
As shown in
It is to be understood that while the invention has been described in conjunction with the detailed description thereof, the foregoing description is intended to illustrate and not limit the scope of the invention, which is defined by the scope of the appended claims. Other aspects, advantages, and modifications are within the scope of the following claims. In case of conflict, the present specification, including definitions, will control. In addition, section headings, the materials, methods, and examples are illustrative only and not intended to be limiting.
This application claims priority to U.S. Provisional Patent Application No. 63/068,146, filed Aug. 20, 2020. The entire content of the foregoing application is incorporated herein by reference.
Number | Name | Date | Kind |
---|---|---|---|
4683195 | Mullis | Jul 1987 | A |
4683202 | Mullis | Jul 1987 | A |
4800159 | Mullis | Jan 1989 | A |
4883867 | Lee | Nov 1989 | A |
4965188 | Mullis | Oct 1990 | A |
4988617 | Landegren et al. | Jan 1991 | A |
5002882 | Lunnen | Mar 1991 | A |
5130238 | Malek | Jul 1992 | A |
5308751 | Ohkawa | May 1994 | A |
5321130 | Yue | Jun 1994 | A |
5410030 | Yue | Apr 1995 | A |
5436134 | Haugland | Jul 1995 | A |
5455166 | Walker | Oct 1995 | A |
5494810 | Barany et al. | Feb 1996 | A |
5503980 | Cantor | Apr 1996 | A |
5512439 | Hornes | Apr 1996 | A |
5512462 | Cheng | Apr 1996 | A |
5582977 | Yue | Dec 1996 | A |
5599675 | Brenner | Feb 1997 | A |
5641658 | Adams | Jun 1997 | A |
5648245 | Fire et al. | Jul 1997 | A |
5658751 | Yue | Aug 1997 | A |
5695940 | Drmanac et al. | Dec 1997 | A |
5750341 | Macevicz | May 1998 | A |
5763175 | Brenner | Jun 1998 | A |
5830711 | Barany et al. | Nov 1998 | A |
5837832 | Chee et al. | Nov 1998 | A |
5854033 | Lizardi | Dec 1998 | A |
5863753 | Haugland | Jan 1999 | A |
5871921 | Landegren et al. | Feb 1999 | A |
5912148 | Eggerding | Jun 1999 | A |
5925545 | Reznikoff et al. | Jul 1999 | A |
5928906 | Koester et al. | Jul 1999 | A |
5958775 | Wickstrrom | Sep 1999 | A |
5965443 | Reznikoff et al. | Oct 1999 | A |
6013440 | Lipshutz | Jan 2000 | A |
6027889 | Barany et al. | Feb 2000 | A |
6054274 | Sampson et al. | Apr 2000 | A |
6060240 | Kamb et al. | May 2000 | A |
6130073 | Eggerding | Oct 2000 | A |
6143496 | Brown | Nov 2000 | A |
6153389 | Haarer | Nov 2000 | A |
6159736 | Reznikoff et al. | Dec 2000 | A |
6165714 | Lane et al. | Dec 2000 | A |
6210891 | Nyren | Apr 2001 | B1 |
6210894 | Brennan | Apr 2001 | B1 |
6214587 | Dattagupta | Apr 2001 | B1 |
6251639 | Kurn | Jun 2001 | B1 |
6258568 | Nyren | Jul 2001 | B1 |
6266459 | Walt | Jul 2001 | B1 |
6268148 | Barany et al. | Jul 2001 | B1 |
6274320 | Rothberg | Aug 2001 | B1 |
6291180 | Chu | Sep 2001 | B1 |
6291187 | Kingsmore et al. | Sep 2001 | B1 |
6300063 | Lipshutz et al. | Oct 2001 | B1 |
6309824 | Drmanac | Oct 2001 | B1 |
6323009 | Lasken et al. | Nov 2001 | B1 |
6344316 | Lockhart | Feb 2002 | B1 |
6344329 | Lizardi et al. | Feb 2002 | B1 |
6355431 | Chee | Mar 2002 | B1 |
6368801 | Faruqi | Apr 2002 | B1 |
6401267 | Drmanac | Jun 2002 | B1 |
6404907 | Gilchrist | Jun 2002 | B1 |
6432360 | Church et al. | Aug 2002 | B1 |
6503713 | Rana | Jan 2003 | B1 |
6506561 | Cheval et al. | Jan 2003 | B1 |
6534266 | Singer | Mar 2003 | B1 |
6544732 | Chee | Apr 2003 | B1 |
6573043 | Cohen et al. | Jun 2003 | B1 |
6579695 | Lambalot | Jun 2003 | B1 |
6620584 | Chee | Sep 2003 | B1 |
6632641 | Brennan | Oct 2003 | B1 |
6737236 | Pieken et al. | May 2004 | B1 |
6770441 | Dickinson | Aug 2004 | B2 |
6773886 | Kaufman | Aug 2004 | B2 |
6787308 | Balasubramanian | Sep 2004 | B2 |
6797470 | Barany et al. | Sep 2004 | B2 |
6800453 | Labaer | Oct 2004 | B2 |
6812005 | Fan et al. | Nov 2004 | B2 |
6828100 | Ronaghi | Dec 2004 | B1 |
6833246 | Balasubramanian | Dec 2004 | B2 |
6852487 | Barany et al. | Feb 2005 | B1 |
6859570 | Walt | Feb 2005 | B2 |
6864052 | Drmanac | Mar 2005 | B1 |
6867028 | Janulaitis | Mar 2005 | B2 |
6872816 | Hall et al. | Mar 2005 | B1 |
6875572 | Prudent et al. | Apr 2005 | B2 |
6890741 | Fan et al. | May 2005 | B2 |
6897023 | Fu | May 2005 | B2 |
6913881 | Aizenstein et al. | Jul 2005 | B1 |
6942968 | Dickinson et al. | Sep 2005 | B1 |
7011944 | Prudent et al. | Mar 2006 | B2 |
7057026 | Barnes | Jun 2006 | B2 |
7083980 | Reznikoff et al. | Aug 2006 | B2 |
7115400 | Adessi | Oct 2006 | B1 |
7118883 | Inoue | Oct 2006 | B2 |
7166431 | Chee et al. | Jan 2007 | B2 |
7192735 | Lambalot | Mar 2007 | B2 |
7211414 | Hardin | May 2007 | B2 |
7255994 | Lao | Aug 2007 | B2 |
7258976 | Mitsuhashi | Aug 2007 | B2 |
7282328 | Kong et al. | Oct 2007 | B2 |
7297518 | Quake | Nov 2007 | B2 |
7329492 | Hardin | Feb 2008 | B2 |
7358047 | Hafner et al. | Apr 2008 | B2 |
7361488 | Fan et al. | Apr 2008 | B2 |
7378242 | Hurt | May 2008 | B2 |
7393665 | Brenner | Jul 2008 | B2 |
7405281 | Xu | Jul 2008 | B2 |
7407757 | Brenner | Aug 2008 | B2 |
7473767 | Dimitrov | Jan 2009 | B2 |
7499806 | Kermani et al. | Mar 2009 | B2 |
7537897 | Brenner | May 2009 | B2 |
7563576 | Chee | Jul 2009 | B2 |
7579153 | Brenner | Aug 2009 | B2 |
7582420 | Oliphant et al. | Sep 2009 | B2 |
7601498 | Mao | Oct 2009 | B2 |
7608434 | Reznikoff et al. | Oct 2009 | B2 |
7611869 | Fan | Nov 2009 | B2 |
7635566 | Brenner | Dec 2009 | B2 |
7666612 | Johnsson | Feb 2010 | B2 |
7674752 | He | Mar 2010 | B2 |
7709198 | Luo et al. | May 2010 | B2 |
7776547 | Roth | Aug 2010 | B2 |
7776567 | Mao | Aug 2010 | B2 |
7803943 | Mao | Sep 2010 | B2 |
7888009 | Barany et al. | Feb 2011 | B2 |
7892747 | Barany et al. | Feb 2011 | B2 |
7910304 | Drmanac | Mar 2011 | B2 |
7914981 | Barany et al. | Mar 2011 | B2 |
7955794 | Shen et al. | Jun 2011 | B2 |
7960119 | Chee | Jun 2011 | B2 |
7985565 | Mayer et al. | Jul 2011 | B2 |
8003354 | Shen et al. | Aug 2011 | B2 |
8076063 | Fan | Dec 2011 | B2 |
8092784 | Mao | Jan 2012 | B2 |
8148068 | Brenner | Apr 2012 | B2 |
8206917 | Chee | Jun 2012 | B2 |
8268554 | Schallmeiner | Sep 2012 | B2 |
8288103 | Oliphant | Oct 2012 | B2 |
8288122 | O'Leary et al. | Oct 2012 | B2 |
8383338 | Kitzman | Feb 2013 | B2 |
8431691 | McKernan et al. | Apr 2013 | B2 |
8460865 | Chee | Jun 2013 | B2 |
8481257 | Van Eijk | Jul 2013 | B2 |
8481258 | Church et al. | Jul 2013 | B2 |
8481292 | Casbon | Jul 2013 | B2 |
8481698 | Lieberman et al. | Jul 2013 | B2 |
8507204 | Pierce et al. | Aug 2013 | B2 |
8519115 | Webster et al. | Aug 2013 | B2 |
8551710 | Bernitz et al. | Oct 2013 | B2 |
8568979 | Stuelpnagel et al. | Oct 2013 | B2 |
8586310 | Mitra | Nov 2013 | B2 |
8597891 | Barany et al. | Dec 2013 | B2 |
8603743 | Liu et al. | Dec 2013 | B2 |
8604182 | Luo et al. | Dec 2013 | B2 |
8614073 | Van Eijk | Dec 2013 | B2 |
8624016 | Barany et al. | Jan 2014 | B2 |
8685889 | Van Eijk | Apr 2014 | B2 |
8741564 | Seligmann | Jun 2014 | B2 |
8741606 | Casbon | Jun 2014 | B2 |
8771950 | Church et al. | Jul 2014 | B2 |
8785353 | Van Eijk | Jul 2014 | B2 |
8790873 | Namsaraev et al. | Jul 2014 | B2 |
8809238 | Livak et al. | Aug 2014 | B2 |
8815512 | Van Eijk | Aug 2014 | B2 |
8835358 | Fodor | Sep 2014 | B2 |
8865410 | Shendure | Oct 2014 | B2 |
8906626 | Oliphant et al. | Dec 2014 | B2 |
8911945 | Van Eijk | Dec 2014 | B2 |
8936912 | Mitra | Jan 2015 | B2 |
8951726 | Luo et al. | Feb 2015 | B2 |
8951728 | Rasmussen | Feb 2015 | B2 |
8986926 | Ferree et al. | Mar 2015 | B2 |
9005891 | Sinicropi et al. | Apr 2015 | B2 |
9005935 | Belyaev | Apr 2015 | B2 |
9023768 | Van Eijk | May 2015 | B2 |
9062348 | Van Eijk | Jun 2015 | B1 |
9080210 | Van Eijk | Jul 2015 | B2 |
9194001 | Brenner | Nov 2015 | B2 |
9201063 | Sood et al. | Dec 2015 | B2 |
9273349 | Nguyen et al. | Mar 2016 | B2 |
9290808 | Fodor | Mar 2016 | B2 |
9290809 | Fodor | Mar 2016 | B2 |
9328383 | Van Eijk | May 2016 | B2 |
9334536 | Van Eijk | May 2016 | B2 |
9371563 | Geiss et al. | Jun 2016 | B2 |
9371598 | Chee | Jun 2016 | B2 |
9376716 | Van Eijk | Jun 2016 | B2 |
9376717 | Gao et al. | Jun 2016 | B2 |
9376719 | Eijk | Jun 2016 | B2 |
9416409 | Hayden | Aug 2016 | B2 |
9447459 | Van Eijk | Sep 2016 | B2 |
9453256 | Van Eijk | Sep 2016 | B2 |
9493820 | Van Eijk | Nov 2016 | B2 |
9506061 | Brown et al. | Nov 2016 | B2 |
9512422 | Barnard et al. | Dec 2016 | B2 |
9574230 | Van Eijk | Feb 2017 | B2 |
9593365 | Frisen et al. | Mar 2017 | B2 |
9598728 | Barany et al. | Mar 2017 | B2 |
9624538 | Church et al. | Apr 2017 | B2 |
9644204 | Hindson et al. | May 2017 | B2 |
9657335 | Van Eijk | May 2017 | B2 |
9670542 | Eijk | Jun 2017 | B2 |
9694361 | Bharadwaj | Jul 2017 | B2 |
9702004 | Van Eijk | Jul 2017 | B2 |
9714446 | Webster et al. | Jul 2017 | B2 |
9714937 | Dunaway | Jul 2017 | B2 |
9727810 | Fodor et al. | Aug 2017 | B2 |
9745627 | Van Eijk | Aug 2017 | B2 |
9777324 | Van Eijk | Oct 2017 | B2 |
9783841 | Nolan et al. | Oct 2017 | B2 |
9790476 | Gloeckner et al. | Oct 2017 | B2 |
9816134 | Namsaraev | Nov 2017 | B2 |
9834814 | Peter et al. | Dec 2017 | B2 |
9850536 | Oliphant et al. | Dec 2017 | B2 |
9856521 | Stevens et al. | Jan 2018 | B2 |
9868979 | Chee et al. | Jan 2018 | B2 |
9879313 | Chee et al. | Jan 2018 | B2 |
9896721 | Van Eijk | Feb 2018 | B2 |
9898576 | Van Eijk | Feb 2018 | B2 |
9898577 | Van Eijk | Feb 2018 | B2 |
9902991 | Sinicropi et al. | Feb 2018 | B2 |
9909167 | Samusik et al. | Mar 2018 | B2 |
9938566 | Shepard et al. | Apr 2018 | B2 |
9957550 | Yeakley et al. | May 2018 | B2 |
10002316 | Fodor et al. | Jun 2018 | B2 |
10023907 | Van Eijk | Jul 2018 | B2 |
10030261 | Frisen et al. | Jul 2018 | B2 |
10035992 | Gloeckner et al. | Jul 2018 | B2 |
10041949 | Bendall et al. | Aug 2018 | B2 |
10059989 | Giresi et al. | Aug 2018 | B2 |
10059990 | Boyden et al. | Aug 2018 | B2 |
10095832 | Van Eijk | Oct 2018 | B2 |
10144966 | Cantor | Dec 2018 | B2 |
10208982 | Bannish et al. | Feb 2019 | B2 |
10227639 | Levner et al. | Mar 2019 | B2 |
10273541 | Hindson et al. | Apr 2019 | B2 |
10357771 | Bharadwaj | Jul 2019 | B2 |
10370698 | Nolan et al. | Aug 2019 | B2 |
10415080 | Dunaway et al. | Sep 2019 | B2 |
10465235 | Gullberg et al. | Nov 2019 | B2 |
10472669 | Chee | Nov 2019 | B2 |
10480022 | Chee | Nov 2019 | B2 |
10480029 | Bent et al. | Nov 2019 | B2 |
10494667 | Chee | Dec 2019 | B2 |
10495554 | Deisseroth et al. | Dec 2019 | B2 |
10501777 | Beechem et al. | Dec 2019 | B2 |
10501791 | Church et al. | Dec 2019 | B2 |
10510435 | Cai et al. | Dec 2019 | B2 |
10544403 | Gloeckner et al. | Jan 2020 | B2 |
10550429 | Harada et al. | Feb 2020 | B2 |
10590244 | Delaney et al. | Mar 2020 | B2 |
10633648 | Seelig et al. | Apr 2020 | B2 |
10640816 | Beechem et al. | May 2020 | B2 |
10640826 | Church et al. | May 2020 | B2 |
10669569 | Gullberg et al. | Jun 2020 | B2 |
10724078 | Van Driel et al. | Jul 2020 | B2 |
10725027 | Bell | Jul 2020 | B2 |
10774372 | Chee et al. | Sep 2020 | B2 |
10774374 | Frisen et al. | Sep 2020 | B2 |
10787701 | Chee | Sep 2020 | B2 |
10815519 | Husain et al. | Oct 2020 | B2 |
10829803 | Terbrueggen et al. | Nov 2020 | B2 |
10844426 | Daugharthy et al. | Nov 2020 | B2 |
10858698 | Church et al. | Dec 2020 | B2 |
10858702 | Lucero et al. | Dec 2020 | B2 |
10913975 | So et al. | Feb 2021 | B2 |
10914730 | Chee et al. | Feb 2021 | B2 |
10927403 | Chee et al. | Feb 2021 | B2 |
10961566 | Chee | Mar 2021 | B2 |
11008607 | Chee | May 2021 | B2 |
11046996 | Chee et al. | Jun 2021 | B1 |
11067567 | Chee | Jul 2021 | B2 |
11104936 | Zhang et al. | Aug 2021 | B2 |
11118216 | Koshinsky et al. | Sep 2021 | B2 |
11156603 | Chee | Oct 2021 | B2 |
11162132 | Frisen et al. | Nov 2021 | B2 |
11208684 | Chee | Dec 2021 | B2 |
11286515 | Chee et al. | Mar 2022 | B2 |
11293917 | Chee | Apr 2022 | B2 |
11299774 | Frisen et al. | Apr 2022 | B2 |
11313856 | Chee | Apr 2022 | B2 |
11332790 | Chell et al. | May 2022 | B2 |
11352659 | Frisen et al. | Jun 2022 | B2 |
11352667 | Hauling et al. | Jun 2022 | B2 |
11359228 | Chee et al. | Jun 2022 | B2 |
11365442 | Chee | Jun 2022 | B2 |
11371086 | Chee | Jun 2022 | B2 |
11384386 | Chee | Jul 2022 | B2 |
11390912 | Frisen et al. | Jul 2022 | B2 |
11401545 | Chee | Aug 2022 | B2 |
11407992 | Dadhwal | Aug 2022 | B2 |
11408029 | Katiraee et al. | Aug 2022 | B2 |
11434524 | Ramachandran Iyer et al. | Sep 2022 | B2 |
11479809 | Frisen et al. | Oct 2022 | B2 |
11479810 | Chee | Oct 2022 | B1 |
11492612 | Dadhwal | Nov 2022 | B1 |
11505828 | Chell et al. | Nov 2022 | B2 |
11512308 | Gallant et al. | Nov 2022 | B2 |
11519022 | Chee | Dec 2022 | B2 |
11519033 | Schnall-Levin et al. | Dec 2022 | B2 |
11530438 | Persson et al. | Dec 2022 | B2 |
11535887 | Gallant et al. | Dec 2022 | B2 |
11542543 | Chee | Jan 2023 | B2 |
11549138 | Chee | Jan 2023 | B2 |
11560587 | Chee | Jan 2023 | B2 |
11560592 | Chew et al. | Jan 2023 | B2 |
11560593 | Chell et al. | Jan 2023 | B2 |
11592447 | Uytingco et al. | Feb 2023 | B2 |
11608498 | Gallant et al. | Mar 2023 | B2 |
11608520 | Galonska | Mar 2023 | B2 |
11613773 | Frisen et al. | Mar 2023 | B2 |
11618897 | Kim et al. | Apr 2023 | B2 |
11618918 | Chee et al. | Apr 2023 | B2 |
11624063 | Dadhwal | Apr 2023 | B2 |
11624086 | Uytingco et al. | Apr 2023 | B2 |
11634756 | Chee | Apr 2023 | B2 |
11649485 | Yin et al. | May 2023 | B2 |
11661626 | Katiraee et al. | May 2023 | B2 |
11680260 | Kim et al. | Jun 2023 | B2 |
11692218 | Engblom et al. | Jul 2023 | B2 |
11702693 | Bharadwaj | Jul 2023 | B2 |
11702698 | Stoeckius | Jul 2023 | B2 |
11732292 | Chee | Aug 2023 | B2 |
11732299 | Ramachandran Iyer | Aug 2023 | B2 |
11732300 | Bava | Aug 2023 | B2 |
11733238 | Chee | Aug 2023 | B2 |
11739372 | Frisen et al. | Aug 2023 | B2 |
11739381 | Chew et al. | Aug 2023 | B2 |
11753673 | Chew et al. | Sep 2023 | B2 |
11753674 | Chee et al. | Sep 2023 | B2 |
11753675 | Ramachandran Iyer | Sep 2023 | B2 |
11761030 | Chee | Sep 2023 | B2 |
11761038 | Stoeckius | Sep 2023 | B1 |
11767550 | Chee | Sep 2023 | B2 |
11768175 | Kim et al. | Sep 2023 | B1 |
11773433 | Gallant et al. | Oct 2023 | B2 |
11781130 | Dadhwal | Oct 2023 | B2 |
11788122 | Frisen et al. | Oct 2023 | B2 |
11795498 | Frisen et al. | Oct 2023 | B2 |
11795507 | Chell et al. | Oct 2023 | B2 |
11808769 | Uytingco et al. | Nov 2023 | B2 |
11821024 | Chee et al. | Nov 2023 | B2 |
11821035 | Bent et al. | Nov 2023 | B1 |
11827935 | Ramachandran Iyer et al. | Nov 2023 | B1 |
11835462 | Bava | Dec 2023 | B2 |
11840687 | Gallant et al. | Dec 2023 | B2 |
11840724 | Chew et al. | Dec 2023 | B2 |
11845979 | Engblom et al. | Dec 2023 | B2 |
11859178 | Gallant et al. | Jan 2024 | B2 |
11866767 | Uytingco et al. | Jan 2024 | B2 |
11866770 | Chee | Jan 2024 | B2 |
11873482 | Kim et al. | Jan 2024 | B2 |
11891654 | Alvarado Martinez et al. | Feb 2024 | B2 |
20010055764 | Empendocles et al. | Dec 2001 | A1 |
20020040275 | Cravatt | Apr 2002 | A1 |
20020051986 | Baez et al. | May 2002 | A1 |
20020055100 | Kawashima | May 2002 | A1 |
20020058250 | Firth | May 2002 | A1 |
20020086441 | Baranov et al. | Jul 2002 | A1 |
20020164611 | Bamdad | Nov 2002 | A1 |
20030017451 | Wang et al. | Jan 2003 | A1 |
20030022207 | Balasubramanian | Jan 2003 | A1 |
20030064398 | Barnes | Apr 2003 | A1 |
20030138879 | Lambalot | Jul 2003 | A1 |
20030148335 | Shen et al. | Aug 2003 | A1 |
20030162216 | Gold | Aug 2003 | A1 |
20030165948 | Alsmadi et al. | Sep 2003 | A1 |
20030211489 | Shen et al. | Nov 2003 | A1 |
20030224419 | Corcoran | Dec 2003 | A1 |
20030232348 | Jones et al. | Dec 2003 | A1 |
20030232382 | Brennan | Dec 2003 | A1 |
20030235854 | Chan et al. | Dec 2003 | A1 |
20040033499 | Ilsley et al. | Feb 2004 | A1 |
20040067492 | Peng et al. | Apr 2004 | A1 |
20040082059 | Webb et al. | Apr 2004 | A1 |
20040096853 | Mayer | May 2004 | A1 |
20040106110 | Balasubramanian | Jun 2004 | A1 |
20040235103 | Reznikoff et al. | Nov 2004 | A1 |
20040248325 | Bukusoglu et al. | Dec 2004 | A1 |
20040259105 | Fan et al. | Dec 2004 | A1 |
20050003431 | Wucherpfennig | Jan 2005 | A1 |
20050014203 | Darfler et al. | Jan 2005 | A1 |
20050037393 | Gunderson et al. | Feb 2005 | A1 |
20050048580 | Labaer | Mar 2005 | A1 |
20050064460 | Holliger et al. | Mar 2005 | A1 |
20050095627 | Kolman et al. | May 2005 | A1 |
20050100900 | Kawashima et al. | May 2005 | A1 |
20050130173 | Leamon et al. | Jun 2005 | A1 |
20050136414 | Gunderson et al. | Jun 2005 | A1 |
20050164292 | Farooqui | Jul 2005 | A1 |
20050191656 | Drmanac et al. | Sep 2005 | A1 |
20050191698 | Chee et al. | Sep 2005 | A1 |
20050202433 | Van Beuningen | Sep 2005 | A1 |
20050227271 | Kwon | Oct 2005 | A1 |
20050239119 | Tsukada et al. | Oct 2005 | A1 |
20050260653 | LaBaer | Nov 2005 | A1 |
20050266417 | Barany et al. | Dec 2005 | A1 |
20060046313 | Roth | Mar 2006 | A1 |
20060084078 | Zhao | Apr 2006 | A1 |
20060105352 | Qiao et al. | May 2006 | A1 |
20060154286 | Kong et al. | Jul 2006 | A1 |
20060188901 | Barnes et al. | Aug 2006 | A1 |
20060199183 | Valat et al. | Sep 2006 | A1 |
20060211001 | Yu et al. | Sep 2006 | A1 |
20060216775 | Burkart et al. | Sep 2006 | A1 |
20060240439 | Smith et al. | Oct 2006 | A1 |
20060263789 | Kincaid | Nov 2006 | A1 |
20060275782 | Gunderson et al. | Dec 2006 | A1 |
20060281109 | Barr Ost et al. | Dec 2006 | A1 |
20070020640 | McCloskey et al. | Jan 2007 | A1 |
20070020669 | Ericsson | Jan 2007 | A1 |
20070026430 | Andersen et al. | Feb 2007 | A1 |
20070054288 | Su et al. | Mar 2007 | A1 |
20070087360 | Boyd | Apr 2007 | A1 |
20070099208 | Drmanac et al. | May 2007 | A1 |
20070128624 | Gormley et al. | Jun 2007 | A1 |
20070128656 | Agrawal | Jun 2007 | A1 |
20070134723 | Kozlov et al. | Jun 2007 | A1 |
20070161020 | Luo et al. | Jul 2007 | A1 |
20070166705 | Milton et al. | Jul 2007 | A1 |
20070172873 | Brenner et al. | Jul 2007 | A1 |
20070207482 | Church et al. | Sep 2007 | A1 |
20070254305 | Paik et al. | Nov 2007 | A1 |
20070269805 | Hogers | Nov 2007 | A1 |
20080003586 | Hyde et al. | Jan 2008 | A1 |
20080009420 | Schroth et al. | Jan 2008 | A1 |
20080108082 | Rank et al. | May 2008 | A1 |
20080108804 | Hayashizaki et al. | May 2008 | A1 |
20080132429 | Perov et al. | Jun 2008 | A1 |
20080160580 | Adessi et al. | Jul 2008 | A1 |
20080220434 | Thomas | Sep 2008 | A1 |
20080261204 | Lexow | Oct 2008 | A1 |
20080286795 | Kawashima et al. | Nov 2008 | A1 |
20080293046 | Allawi et al. | Nov 2008 | A1 |
20090005252 | Drmanac et al. | Jan 2009 | A1 |
20090006002 | Honisch et al. | Jan 2009 | A1 |
20090018024 | Church et al. | Jan 2009 | A1 |
20090026082 | Rothberg et al. | Jan 2009 | A1 |
20090036323 | van Eijk et al. | Feb 2009 | A1 |
20090082212 | Williams | Mar 2009 | A1 |
20090099041 | Church et al. | Apr 2009 | A1 |
20090105959 | Braverman et al. | Apr 2009 | A1 |
20090117573 | Fu et al. | May 2009 | A1 |
20090127589 | Rothberg et al. | May 2009 | A1 |
20090155781 | Drmanac et al. | Jun 2009 | A1 |
20090170713 | van Eijk et al. | Jul 2009 | A1 |
20090202998 | Schlumpberger et al. | Aug 2009 | A1 |
20090233802 | Bignell et al. | Sep 2009 | A1 |
20090253581 | van Eijk et al. | Oct 2009 | A1 |
20090283407 | Van Eijk | Nov 2009 | A1 |
20090291854 | Weisinger-Mayr et al. | Nov 2009 | A1 |
20090312193 | Kim et al. | Dec 2009 | A1 |
20100035249 | Hayashizaki et al. | Feb 2010 | A1 |
20100069263 | Shendure et al. | Mar 2010 | A1 |
20100105052 | Drmanac et al. | Apr 2010 | A1 |
20100120097 | Matz et al. | May 2010 | A1 |
20100120098 | Grunenwald et al. | May 2010 | A1 |
20100129874 | Mitra et al. | May 2010 | A1 |
20100145037 | Brive et al. | Jun 2010 | A1 |
20100173384 | Johnsson et al. | Jul 2010 | A1 |
20100184618 | Namsaraev et al. | Jul 2010 | A1 |
20100210475 | Lee et al. | Aug 2010 | A1 |
20100227329 | Cuppens | Sep 2010 | A1 |
20100273219 | May et al. | Oct 2010 | A1 |
20110028685 | Purkayastha et al. | Feb 2011 | A1 |
20110033854 | Drmanac et al. | Feb 2011 | A1 |
20110045462 | Fu et al. | Feb 2011 | A1 |
20110059436 | Hardin et al. | Mar 2011 | A1 |
20110111409 | Sinicropi et al. | May 2011 | A1 |
20110152111 | Fan et al. | Jun 2011 | A1 |
20110245101 | Chee et al. | Oct 2011 | A1 |
20110245111 | Chee | Oct 2011 | A1 |
20110287435 | Grunenwald et al. | Nov 2011 | A1 |
20120021930 | Schoen et al. | Jan 2012 | A1 |
20120046175 | Rodesch et al. | Feb 2012 | A1 |
20120046178 | Van Den Boom et al. | Feb 2012 | A1 |
20120065081 | Chee | Mar 2012 | A1 |
20120135871 | van Eijk et al. | May 2012 | A1 |
20120202698 | van Eijk et al. | Aug 2012 | A1 |
20120202704 | Fan et al. | Aug 2012 | A1 |
20120220479 | Ericsson et al. | Aug 2012 | A1 |
20120245053 | Shirai et al. | Sep 2012 | A1 |
20120252702 | Muratani et al. | Oct 2012 | A1 |
20120258871 | Kozlov et al. | Oct 2012 | A1 |
20120289414 | Mitra et al. | Nov 2012 | A1 |
20120301925 | Belyaev | Nov 2012 | A1 |
20130005594 | Terbrueggen et al. | Jan 2013 | A1 |
20130005600 | Olek | Jan 2013 | A1 |
20130023433 | Luo et al. | Jan 2013 | A1 |
20130035239 | Kong et al. | Feb 2013 | A1 |
20130065768 | Zheng et al. | Mar 2013 | A1 |
20130079232 | Kain et al. | Mar 2013 | A1 |
20130171621 | Luo et al. | Jul 2013 | A1 |
20130244884 | Jacobson et al. | Sep 2013 | A1 |
20130261019 | Lin et al. | Oct 2013 | A1 |
20130302801 | Asbury et al. | Nov 2013 | A1 |
20130338042 | Shen et al. | Dec 2013 | A1 |
20140066318 | Frisen et al. | Mar 2014 | A1 |
20140121118 | Warner | May 2014 | A1 |
20140270435 | Dunn | Sep 2014 | A1 |
20140274731 | Raymond et al. | Sep 2014 | A1 |
20140323330 | Glezer et al. | Oct 2014 | A1 |
20140342921 | Weiner | Nov 2014 | A1 |
20140378350 | Hindson et al. | Dec 2014 | A1 |
20150000854 | Gann-Fetter et al. | Jan 2015 | A1 |
20150292988 | Bharadwaj et al. | Oct 2015 | A1 |
20150344942 | Frisen et al. | Dec 2015 | A1 |
20160019337 | Roberts et al. | Jan 2016 | A1 |
20160024576 | Chee | Jan 2016 | A1 |
20160041159 | Labaer et al. | Feb 2016 | A1 |
20160060687 | Zhu et al. | Mar 2016 | A1 |
20160108458 | Frei et al. | Apr 2016 | A1 |
20160122817 | Jarosz et al. | May 2016 | A1 |
20160138091 | Chee et al. | May 2016 | A1 |
20160145677 | Chee et al. | May 2016 | A1 |
20160194692 | Gore et al. | Jul 2016 | A1 |
20160201125 | Samuels et al. | Jul 2016 | A1 |
20160253584 | Fodor et al. | Sep 2016 | A1 |
20160289740 | Fu et al. | Oct 2016 | A1 |
20160298180 | Chee | Oct 2016 | A1 |
20160305856 | Boyden et al. | Oct 2016 | A1 |
20160333403 | Chee | Nov 2016 | A1 |
20160376642 | Landegren et al. | Dec 2016 | A1 |
20170009278 | Söderberg et al. | Jan 2017 | A1 |
20170016053 | Beechem et al. | Jan 2017 | A1 |
20170029875 | Zhang et al. | Feb 2017 | A1 |
20170058339 | Chee | Mar 2017 | A1 |
20170058340 | Chee | Mar 2017 | A1 |
20170058345 | Chee | Mar 2017 | A1 |
20170067096 | Wassie et al. | Mar 2017 | A1 |
20170088881 | Chee | Mar 2017 | A1 |
20170089811 | Tillberg et al. | Mar 2017 | A1 |
20170159109 | Zheng et al. | Jun 2017 | A1 |
20170166962 | van Eijk et al. | Jun 2017 | A1 |
20170220733 | Zhuang et al. | Aug 2017 | A1 |
20170233722 | Seelig et al. | Aug 2017 | A1 |
20170241911 | Rockel et al. | Aug 2017 | A1 |
20170283860 | Kool et al. | Oct 2017 | A1 |
20170335297 | Ha et al. | Nov 2017 | A1 |
20170335410 | Driscoll et al. | Nov 2017 | A1 |
20170342405 | Fu et al. | Nov 2017 | A1 |
20170349940 | Morin et al. | Dec 2017 | A1 |
20180051322 | Church et al. | Feb 2018 | A1 |
20180057873 | Zhou et al. | Mar 2018 | A1 |
20180080019 | Blainey et al. | Mar 2018 | A1 |
20180094316 | Oliphant et al. | Apr 2018 | A1 |
20180105808 | Mikkelsen et al. | Apr 2018 | A1 |
20180112261 | Van Driel et al. | Apr 2018 | A1 |
20180127817 | Borchert et al. | May 2018 | A1 |
20180163265 | Zhang et al. | Jun 2018 | A1 |
20180179591 | van Eijk | Jun 2018 | A1 |
20180201925 | Steemers et al. | Jul 2018 | A1 |
20180201980 | Chee et al. | Jul 2018 | A1 |
20180208967 | Larman et al. | Jul 2018 | A1 |
20180216161 | Chen et al. | Aug 2018 | A1 |
20180216162 | Belhocine et al. | Aug 2018 | A1 |
20180237864 | Imler et al. | Aug 2018 | A1 |
20180245142 | So et al. | Aug 2018 | A1 |
20180247017 | van Eijk et al. | Aug 2018 | A1 |
20180291427 | Edelman | Oct 2018 | A1 |
20180291439 | van Eijk et al. | Oct 2018 | A1 |
20180305681 | Jovanovich et al. | Oct 2018 | A1 |
20180312822 | Lee et al. | Nov 2018 | A1 |
20180320226 | Church et al. | Nov 2018 | A1 |
20190055594 | Samusik et al. | Feb 2019 | A1 |
20190064173 | Bharadwaj et al. | Feb 2019 | A1 |
20190071656 | Chang et al. | Mar 2019 | A1 |
20190085383 | Church et al. | Mar 2019 | A1 |
20190119735 | Deisseroth et al. | Apr 2019 | A1 |
20190135774 | Orbai | May 2019 | A1 |
20190145982 | Chee et al. | May 2019 | A1 |
20190161796 | Hauling et al. | May 2019 | A1 |
20190177777 | Chee | Jun 2019 | A1 |
20190177778 | Chee | Jun 2019 | A1 |
20190177789 | Hindson et al. | Jun 2019 | A1 |
20190177800 | Boutet et al. | Jun 2019 | A1 |
20190194709 | Church et al. | Jun 2019 | A1 |
20190203275 | Frisen et al. | Jul 2019 | A1 |
20190218276 | Regev et al. | Jul 2019 | A1 |
20190218608 | Daugharthy et al. | Jul 2019 | A1 |
20190233878 | Delaney et al. | Aug 2019 | A1 |
20190233880 | Mir | Aug 2019 | A1 |
20190249226 | Bent et al. | Aug 2019 | A1 |
20190262831 | West et al. | Aug 2019 | A1 |
20190264268 | Frisen et al. | Aug 2019 | A1 |
20190271028 | Khafizov et al. | Sep 2019 | A1 |
20190271030 | Chee | Sep 2019 | A1 |
20190271031 | Chee | Sep 2019 | A1 |
20190300943 | Chee et al. | Oct 2019 | A1 |
20190300944 | Chee et al. | Oct 2019 | A1 |
20190300945 | Chee et al. | Oct 2019 | A1 |
20190309353 | Chee | Oct 2019 | A1 |
20190309354 | Chee | Oct 2019 | A1 |
20190309355 | Chee | Oct 2019 | A1 |
20190323071 | Chee | Oct 2019 | A1 |
20190323088 | Boutet et al. | Oct 2019 | A1 |
20190330617 | Church et al. | Oct 2019 | A1 |
20190338353 | Belgrader et al. | Nov 2019 | A1 |
20190360034 | Zhou et al. | Nov 2019 | A1 |
20190360043 | Pham et al. | Nov 2019 | A1 |
20190367969 | Belhocine et al. | Dec 2019 | A1 |
20190367982 | Belhocine et al. | Dec 2019 | A1 |
20190367997 | Bent et al. | Dec 2019 | A1 |
20200002763 | Belgrader et al. | Jan 2020 | A1 |
20200010891 | Beechem et al. | Jan 2020 | A1 |
20200024641 | Nolan et al. | Jan 2020 | A1 |
20200047010 | Lee et al. | Feb 2020 | A1 |
20200048690 | Chee | Feb 2020 | A1 |
20200063191 | Kennedy-Darling et al. | Feb 2020 | A1 |
20200063195 | Chee | Feb 2020 | A1 |
20200063196 | Chee | Feb 2020 | A1 |
20200071751 | Daugharthy et al. | Mar 2020 | A1 |
20200080136 | Zhang et al. | Mar 2020 | A1 |
20200109443 | Chee | Apr 2020 | A1 |
20200123597 | Daniel | Apr 2020 | A1 |
20200140920 | Pierce et al. | May 2020 | A1 |
20200173985 | Dong et al. | Jun 2020 | A1 |
20200199565 | Chen et al. | Jun 2020 | A1 |
20200199572 | Kuersten et al. | Jun 2020 | A1 |
20200224244 | Nilsson et al. | Jul 2020 | A1 |
20200239874 | Mikkelsen | Jul 2020 | A1 |
20200256867 | Hennek et al. | Aug 2020 | A1 |
20200277663 | Iyer | Sep 2020 | A1 |
20200277664 | Frenz | Sep 2020 | A1 |
20200283852 | Oliphant et al. | Sep 2020 | A1 |
20200299757 | Chee et al. | Sep 2020 | A1 |
20200325531 | Chee | Oct 2020 | A1 |
20200362398 | Kishi et al. | Nov 2020 | A1 |
20200370095 | Farmer et al. | Nov 2020 | A1 |
20200399687 | Frisen et al. | Dec 2020 | A1 |
20200407781 | Schnall-Levin | Dec 2020 | A1 |
20210010068 | Chee et al. | Jan 2021 | A1 |
20210010070 | Schnall-Levin et al. | Jan 2021 | A1 |
20210017587 | Cai et al. | Jan 2021 | A1 |
20210095331 | Fan et al. | Apr 2021 | A1 |
20210115504 | Cai et al. | Apr 2021 | A1 |
20210123040 | Macosko et al. | Apr 2021 | A1 |
20210140982 | Uytingco et al. | May 2021 | A1 |
20210155982 | Yin et al. | May 2021 | A1 |
20210158522 | Weisenfeld et al. | May 2021 | A1 |
20210172007 | Chee et al. | Jun 2021 | A1 |
20210189475 | Tentori et al. | Jun 2021 | A1 |
20210190770 | Delaney et al. | Jun 2021 | A1 |
20210198741 | Williams | Jul 2021 | A1 |
20210199660 | Williams et al. | Jul 2021 | A1 |
20210207202 | Chee | Jul 2021 | A1 |
20210214785 | Stoeckius | Jul 2021 | A1 |
20210222235 | Chee | Jul 2021 | A1 |
20210222241 | Bharadwaj | Jul 2021 | A1 |
20210222242 | Ramachandran Iyer | Jul 2021 | A1 |
20210222253 | Uytingco | Jul 2021 | A1 |
20210223227 | Stoeckius | Jul 2021 | A1 |
20210230584 | Mikkelsen et al. | Jul 2021 | A1 |
20210230681 | Patterson et al. | Jul 2021 | A1 |
20210230692 | Daugharthy et al. | Jul 2021 | A1 |
20210237022 | Bava | Aug 2021 | A1 |
20210238581 | Mikkelsen et al. | Aug 2021 | A1 |
20210238664 | Bava et al. | Aug 2021 | A1 |
20210238675 | Bava | Aug 2021 | A1 |
20210238680 | Bava | Aug 2021 | A1 |
20210247316 | Bava | Aug 2021 | A1 |
20210255175 | Chee et al. | Aug 2021 | A1 |
20210262018 | Bava et al. | Aug 2021 | A1 |
20210262019 | Alvarado Martinez et al. | Aug 2021 | A1 |
20210269864 | Chee | Sep 2021 | A1 |
20210270822 | Chee | Sep 2021 | A1 |
20210285036 | Yin et al. | Sep 2021 | A1 |
20210285046 | Chell et al. | Sep 2021 | A1 |
20210292748 | Frisen et al. | Sep 2021 | A1 |
20210292822 | Frisen et al. | Sep 2021 | A1 |
20210317510 | Chee et al. | Oct 2021 | A1 |
20210317524 | Lucero et al. | Oct 2021 | A1 |
20210324457 | Ramachandran Iyer et al. | Oct 2021 | A1 |
20210332424 | Schnall-Levin | Oct 2021 | A1 |
20210332425 | Pfeiffer et al. | Oct 2021 | A1 |
20210348221 | Chell et al. | Nov 2021 | A1 |
20220002791 | Frisen et al. | Jan 2022 | A1 |
20220003755 | Chee | Jan 2022 | A1 |
20220010367 | Ramachandran Iyer et al. | Jan 2022 | A1 |
20220017951 | Ramachandran Iyer et al. | Jan 2022 | A1 |
20220025446 | Shah | Jan 2022 | A1 |
20220025447 | Tentori et al. | Jan 2022 | A1 |
20220033888 | Schnall-Levin et al. | Feb 2022 | A1 |
20220049293 | Frenz et al. | Feb 2022 | A1 |
20220064630 | Bent et al. | Mar 2022 | A1 |
20220081728 | Williams | Mar 2022 | A1 |
20220090058 | Frisen et al. | Mar 2022 | A1 |
20220090175 | Uytingco et al. | Mar 2022 | A1 |
20220090181 | Gallant et al. | Mar 2022 | A1 |
20220098576 | Dadhwal | Mar 2022 | A1 |
20220098661 | Chew et al. | Mar 2022 | A1 |
20220106632 | Galonska et al. | Apr 2022 | A1 |
20220106633 | Engblom et al. | Apr 2022 | A1 |
20220112486 | Ramachandran Iyer et al. | Apr 2022 | A1 |
20220112545 | Chee | Apr 2022 | A1 |
20220119869 | Ramachandran Iyer et al. | Apr 2022 | A1 |
20220127659 | Frisen et al. | Apr 2022 | A1 |
20220127666 | Katiraee et al. | Apr 2022 | A1 |
20220127672 | Stoeckius | Apr 2022 | A1 |
20220145361 | Frenz et al. | May 2022 | A1 |
20220154255 | Chee et al. | May 2022 | A1 |
20220170083 | Khaled et al. | Jun 2022 | A1 |
20220195422 | Gallant et al. | Jun 2022 | A1 |
20220195505 | Frisen et al. | Jun 2022 | A1 |
20220196644 | Chee | Jun 2022 | A1 |
20220213526 | Frisen et al. | Jul 2022 | A1 |
20220241780 | Tentori et al. | Aug 2022 | A1 |
20220267844 | Ramachandran Iyer et al. | Aug 2022 | A1 |
20220282329 | Chell et al. | Sep 2022 | A1 |
20220290217 | Frenz et al. | Sep 2022 | A1 |
20220290219 | Chee | Sep 2022 | A1 |
20220298560 | Frisen et al. | Sep 2022 | A1 |
20220325325 | Chee et al. | Oct 2022 | A1 |
20220326251 | Uytingco et al. | Oct 2022 | A1 |
20220333171 | Chee | Oct 2022 | A1 |
20220333191 | Mikkelsen et al. | Oct 2022 | A1 |
20220333192 | Uytingco | Oct 2022 | A1 |
20220333195 | Schnall-Levin et al. | Oct 2022 | A1 |
20220334031 | Delaney et al. | Oct 2022 | A1 |
20220348905 | Dadhwal | Nov 2022 | A1 |
20220348992 | Stoeckius et al. | Nov 2022 | A1 |
20220356464 | Kim et al. | Nov 2022 | A1 |
20220364163 | Stahl et al. | Nov 2022 | A1 |
20220389491 | Chee | Dec 2022 | A1 |
20220389503 | Mikkelsen et al. | Dec 2022 | A1 |
20220389504 | Chew et al. | Dec 2022 | A1 |
20220403455 | Ramachandran Iyer et al. | Dec 2022 | A1 |
20220404245 | Chell et al. | Dec 2022 | A1 |
20230002812 | Stoeckius et al. | Jan 2023 | A1 |
20230014008 | Shastry | Jan 2023 | A1 |
20230416807 | Chee | Jan 2023 | A1 |
20230416808 | Sukovich et al. | Jan 2023 | A1 |
20230033960 | Gallant et al. | Feb 2023 | A1 |
20230034039 | Shahjamali | Feb 2023 | A1 |
20230034216 | Bava | Feb 2023 | A1 |
20230040363 | Chee | Feb 2023 | A1 |
20230042088 | Chee | Feb 2023 | A1 |
20230042817 | Mignardi | Feb 2023 | A1 |
20230047782 | Tentori et al. | Feb 2023 | A1 |
20230056549 | Dadhwal | Feb 2023 | A1 |
20230064372 | Chell et al. | Mar 2023 | A1 |
20230069046 | Chew et al. | Mar 2023 | A1 |
20230077364 | Patterson et al. | Mar 2023 | A1 |
20230080543 | Katiraee et al. | Mar 2023 | A1 |
20230081381 | Chew et al. | Mar 2023 | A1 |
20230100497 | Frisen et al. | Mar 2023 | A1 |
20230107023 | Chee | Apr 2023 | A1 |
20230111225 | Chew et al. | Apr 2023 | A1 |
20230113230 | Kim et al. | Apr 2023 | A1 |
20230126825 | Nagendran et al. | Apr 2023 | A1 |
20230129552 | Ramachandran Iyer | Apr 2023 | A1 |
20230135010 | Tentori et al. | May 2023 | A1 |
20230143569 | Iyer et al. | May 2023 | A1 |
20230145575 | Gallant et al. | May 2023 | A1 |
20230147726 | Hadrup et al. | May 2023 | A1 |
20230151412 | Chee | May 2023 | A1 |
20230159994 | Chee | May 2023 | A1 |
20230159995 | Iyer et al. | May 2023 | A1 |
20230160008 | Chell et al. | May 2023 | A1 |
20230175045 | Katsori et al. | Jun 2023 | A1 |
20230183785 | Frisen et al. | Jun 2023 | A1 |
20230194469 | Tentori et al. | Jun 2023 | A1 |
20230194470 | Kim et al. | Jun 2023 | A1 |
20230203478 | Kim et al. | Jun 2023 | A1 |
20230183684 | Gallant et al. | Jul 2023 | A1 |
20230212650 | Chew et al. | Jul 2023 | A1 |
20230212655 | Chee | Jul 2023 | A1 |
20230220368 | Kim | Jul 2023 | A1 |
20230220454 | Bent et al. | Jul 2023 | A1 |
20230220455 | Galonska | Jul 2023 | A1 |
20230227811 | Dadhwal | Jul 2023 | A1 |
20230228762 | Uytingco et al. | Jul 2023 | A1 |
20230242973 | Frisen et al. | Aug 2023 | A1 |
20230242976 | Tentori et al. | Aug 2023 | A1 |
20230265488 | Gohil et al. | Aug 2023 | A1 |
20230265489 | Uytingco et al. | Aug 2023 | A1 |
20230265491 | Tentori et al. | Aug 2023 | A1 |
20230279474 | Katiraee | Sep 2023 | A1 |
20230279477 | Kvastad et al. | Sep 2023 | A1 |
20230279481 | Marrache et al. | Sep 2023 | A1 |
20230287399 | Gallant et al. | Sep 2023 | A1 |
20230287475 | Chell et al. | Sep 2023 | A1 |
20230287481 | Katsori et al. | Sep 2023 | A1 |
20230295699 | Sukovich et al. | Sep 2023 | A1 |
20230295722 | Bharadwaj | Sep 2023 | A1 |
20230304074 | Chee et al. | Sep 2023 | A1 |
20230304078 | Frisen et al. | Sep 2023 | A1 |
20230313279 | Giacomello et al. | Oct 2023 | A1 |
20230323340 | Dadhwal | Oct 2023 | A1 |
20230323434 | Yin et al. | Oct 2023 | A1 |
20230323436 | Chee | Oct 2023 | A1 |
20230323447 | Schnall-Levin et al. | Oct 2023 | A1 |
20230323453 | Stoeckius | Oct 2023 | A1 |
20230332138 | Kim et al. | Oct 2023 | A1 |
20230332211 | Chee | Oct 2023 | A1 |
20230332212 | Chew et al. | Oct 2023 | A1 |
20230332227 | Ramachandran Iyer | Oct 2023 | A1 |
20230332247 | Singh et al. | Oct 2023 | A1 |
20230358733 | Chee | Nov 2023 | A1 |
20230366008 | Chew et al. | Nov 2023 | A1 |
20230383285 | Kim et al. | Nov 2023 | A1 |
20230383344 | Stoeckius | Nov 2023 | A1 |
20230392204 | Chell et al. | Dec 2023 | A1 |
20230393071 | Bava | Dec 2023 | A1 |
20230407404 | Baumgartner et al. | Dec 2023 | A1 |
20230416850 | Singh et al. | Dec 2023 | A1 |
20240002931 | Bava | Jan 2024 | A1 |
20240011081 | Chee | Jan 2024 | A1 |
20240011090 | Chew et al. | Jan 2024 | A1 |
20240018572 | Mignardi | Jan 2024 | A1 |
20240018575 | Gallant et al. | Jan 2024 | A1 |
20240018589 | Schnall-Levin et al. | Jan 2024 | A1 |
20240026445 | Ramachandran Iyer et al. | Jan 2024 | A1 |
20240035937 | Cox et al. | Feb 2024 | A1 |
Number | Date | Country |
---|---|---|
2003200718 | Oct 2006 | AU |
1273609 | Nov 2000 | CN |
1537953 | Oct 2004 | CN |
1680604 | Oct 2005 | CN |
1749752 | Mar 2006 | CN |
1898398 | Jan 2007 | CN |
101142325 | Mar 2008 | CN |
101221182 | Jul 2008 | CN |
101522915 | Sep 2009 | CN |
107849606 | Mar 2018 | CN |
108949924 | Dec 2018 | CN |
1782737 | May 2007 | EP |
1910562 | Apr 2008 | EP |
1923471 | May 2008 | EP |
1929039 | Jun 2008 | EP |
2002017 | Dec 2008 | EP |
2292788 | Mar 2011 | EP |
2302070 | Mar 2011 | EP |
2580351 | Apr 2013 | EP |
2881465 | Jun 2015 | EP |
3013984 | May 2016 | EP |
3511423 | Jul 2019 | EP |
3541956 | Sep 2019 | EP |
2520765 | Jun 2015 | GB |
2007-014297 | Jan 2007 | JP |
2007-074967 | Mar 2007 | JP |
2009-036694 | Feb 2009 | JP |
WO 1989010977 | Nov 1989 | WO |
WO 1991006678 | May 1991 | WO |
WO 1993004199 | Mar 1993 | WO |
WO 1995023875 | Sep 1995 | WO |
WO 1995025116 | Sep 1995 | WO |
WO 1995035505 | Dec 1995 | WO |
WO 1997031256 | Aug 1997 | WO |
WO 1998044151 | Oct 1998 | WO |
WO 2000017390 | Mar 2000 | WO |
WO 2000063437 | Oct 2000 | WO |
WO 2001006012 | Jan 2001 | WO |
WO 2001009363 | Feb 2001 | WO |
WO 2001012862 | Feb 2001 | WO |
WO 2001042796 | Jun 2001 | WO |
WO 2001046402 | Jun 2001 | WO |
WO 2001059161 | Aug 2001 | WO |
WO 2001090415 | Nov 2001 | WO |
WO 2001096608 | Dec 2001 | WO |
WO 2002040874 | May 2002 | WO |
WO 2002059355 | Aug 2002 | WO |
WO 2002059364 | Aug 2002 | WO |
WO 2002077283 | Oct 2002 | WO |
WO 2003002979 | Jan 2003 | WO |
WO 2003008538 | Jan 2003 | WO |
WO 2003010176 | Feb 2003 | WO |
WO 2003102233 | Dec 2003 | WO |
WO 2004015080 | Feb 2004 | WO |
WO 2004067759 | Aug 2004 | WO |
WO 2004081225 | Sep 2004 | WO |
WO 2005007814 | Jan 2005 | WO |
WO 2005010145 | Feb 2005 | WO |
WO 2005026387 | Mar 2005 | WO |
WO 2005042759 | May 2005 | WO |
WO 2005113804 | Dec 2005 | WO |
WO 2006020515 | Feb 2006 | WO |
WO 2006124771 | Nov 2006 | WO |
WO 2006137733 | Dec 2006 | WO |
WO 2007037678 | Apr 2007 | WO |
WO 2007041689 | Apr 2007 | WO |
WO 2007060599 | May 2007 | WO |
WO 2007073171 | Jun 2007 | WO |
WO 2007076726 | Jul 2007 | WO |
WO 2007139766 | Dec 2007 | WO |
WO 2007145612 | Dec 2007 | WO |
WO 2008069906 | Jun 2008 | WO |
WO 2008093098 | Aug 2008 | WO |
WO 2009032167 | Mar 2009 | WO |
WO 2009036525 | Mar 2009 | WO |
WO 2009152928 | Dec 2009 | WO |
WO 2010019826 | Feb 2010 | WO |
WO 2010027870 | Mar 2010 | WO |
WO 2010126614 | Nov 2010 | WO |
WO 2010127186 | Nov 2010 | WO |
WO 2011008502 | Jan 2011 | WO |
WO 2011062933 | May 2011 | WO |
WO 2011068088 | Jun 2011 | WO |
WO 2011127006 | Oct 2011 | WO |
WO 2011155833 | Dec 2011 | WO |
WO 2012049316 | Apr 2012 | WO |
WO 2012061832 | May 2012 | WO |
WO 2012071428 | May 2012 | WO |
WO 2012129242 | Sep 2012 | WO |
WO 2012159089 | Nov 2012 | WO |
WO 2013123442 | Aug 2013 | WO |
WO 2013131962 | Sep 2013 | WO |
WO 2013138510 | Sep 2013 | WO |
WO 2013142389 | Sep 2013 | WO |
WO 2013150082 | Oct 2013 | WO |
WO 2013150083 | Oct 2013 | WO |
WO 2014044724 | Mar 2014 | WO |
WO 2014060483 | Apr 2014 | WO |
WO 2014071361 | May 2014 | WO |
WO 2014130576 | Aug 2014 | WO |
WO 2014144713 | Sep 2014 | WO |
WO 2014152397 | Sep 2014 | WO |
WO 2014210223 | Dec 2014 | WO |
WO 2014210353 | Dec 2014 | WO |
WO 2015031691 | Mar 2015 | WO |
WO 2015069374 | May 2015 | WO |
WO 2015161173 | Oct 2015 | WO |
WO 2016077763 | May 2016 | WO |
WO 2016138496 | Sep 2016 | WO |
WO 2016138500 | Sep 2016 | WO |
WO 2016162309 | Oct 2016 | WO |
WO 2016166128 | Oct 2016 | WO |
WO 2016168825 | Oct 2016 | WO |
WO 2016172362 | Oct 2016 | WO |
WO 2017019456 | Feb 2017 | WO |
WO 2017019481 | Feb 2017 | WO |
WO 2017075293 | May 2017 | WO |
WO 2017096158 | Jul 2017 | WO |
WO 2017143155 | Aug 2017 | WO |
WO 2017156336 | Sep 2017 | WO |
WO 2017184984 | Oct 2017 | WO |
WO 2017192633 | Nov 2017 | WO |
WO 2018023068 | Feb 2018 | WO |
WO 2018026873 | Feb 2018 | WO |
WO 2018045181 | Mar 2018 | WO |
WO 2018064640 | Apr 2018 | WO |
WO 2018085599 | May 2018 | WO |
WO 2018089550 | May 2018 | WO |
WO 2018091676 | May 2018 | WO |
WO 2018136397 | Jul 2018 | WO |
WO 2018136856 | Jul 2018 | WO |
WO 2018144582 | Aug 2018 | WO |
WO 2018175779 | Sep 2018 | WO |
WO 2018209398 | Nov 2018 | WO |
WO 2019023214 | Jan 2019 | WO |
WO 2019032760 | Feb 2019 | WO |
WO 2019068880 | Apr 2019 | WO |
WO 2019113457 | Jun 2019 | WO |
WO 2019126313 | Jun 2019 | WO |
WO 2019140201 | Jul 2019 | WO |
WO 2019165318 | Aug 2019 | WO |
WO 2019213254 | Nov 2019 | WO |
WO 2019213294 | Nov 2019 | WO |
WO 2019241290 | Dec 2019 | WO |
WO 2020028194 | Feb 2020 | WO |
WO 2020047002 | Mar 2020 | WO |
WO 2020047010 | Mar 2020 | WO |
WO 2020053655 | Mar 2020 | WO |
WO 2020056381 | Mar 2020 | WO |
WO 2020061064 | Mar 2020 | WO |
WO 2020061066 | Mar 2020 | WO |
WO 2020061108 | Mar 2020 | WO |
WO 2020076979 | Apr 2020 | WO |
WO 2020099640 | May 2020 | WO |
WO 2020112604 | Jun 2020 | WO |
WO 2020117914 | Jun 2020 | WO |
WO 2020123301 | Jun 2020 | WO |
WO 2020123305 | Jun 2020 | WO |
WO 2020123311 | Jun 2020 | WO |
WO 2020123316 | Jun 2020 | WO |
WO 2020123317 | Jun 2020 | WO |
WO 2020123318 | Jun 2020 | WO |
WO 2020123319 | Jun 2020 | WO |
WO 2020123320 | Jul 2020 | WO |
WO 2020160044 | Aug 2020 | WO |
WO 2020167862 | Aug 2020 | WO |
WO 2020176788 | Sep 2020 | WO |
WO 2020176882 | Sep 2020 | WO |
WO 2020190509 | Sep 2020 | WO |
WO 2020198071 | Oct 2020 | WO |
WO 2020206285 | Oct 2020 | WO |
WO 2020240025 | Dec 2020 | WO |
WO 2020243579 | Dec 2020 | WO |
WO 2020254519 | Dec 2020 | WO |
WO 2021041974 | Mar 2021 | WO |
WO 2021067246 | Apr 2021 | WO |
WO 2021067514 | Apr 2021 | WO |
WO 2021091611 | May 2021 | WO |
WO 2021092433 | May 2021 | WO |
WO 2021097255 | May 2021 | WO |
WO 2021102003 | May 2021 | WO |
WO 2021102005 | May 2021 | WO |
WO 2021102039 | May 2021 | WO |
WO 2021116715 | Jun 2021 | WO |
WO 2021119320 | Jun 2021 | WO |
WO 2021133842 | Jul 2021 | WO |
WO 2021133845 | Jul 2021 | WO |
WO 2021133849 | Jul 2021 | WO |
WO 2021142233 | Jul 2021 | WO |
WO 2021168261 | Aug 2021 | WO |
WO 2021168278 | Aug 2021 | WO |
WO 2021207610 | Oct 2021 | WO |
WO 2021216708 | Oct 2021 | WO |
WO 2021225900 | Nov 2021 | WO |
WO 2021236625 | Nov 2021 | WO |
WO 2021236929 | Nov 2021 | WO |
WO 2021237056 | Nov 2021 | WO |
WO 2021237087 | Nov 2021 | WO |
WO 2021242834 | Dec 2021 | WO |
WO 2021247543 | Dec 2021 | WO |
WO 2021247568 | Dec 2021 | WO |
WO 2021252499 | Dec 2021 | WO |
WO 2021252576 | Dec 2021 | WO |
WO 2021252591 | Dec 2021 | WO |
WO 2021252747 | Dec 2021 | WO |
WO 2021263111 | Dec 2021 | WO |
WO 2022025965 | Feb 2022 | WO |
WO 2022060798 | Mar 2022 | WO |
WO 2022060953 | Mar 2022 | WO |
WO 2022061152 | Mar 2022 | WO |
WO 2022087273 | Apr 2022 | WO |
WO 2022099037 | May 2022 | WO |
WO 2022103712 | May 2022 | WO |
WO 2022109181 | May 2022 | WO |
WO 2022132645 | Jun 2022 | WO |
WO 2022140028 | Jun 2022 | WO |
WO 2022147005 | Jul 2022 | WO |
WO 2022147296 | Jul 2022 | WO |
WO 2022164615 | Aug 2022 | WO |
WO 2022178267 | Aug 2022 | WO |
WO 2022198068 | Sep 2022 | WO |
WO 2022212269 | Oct 2022 | WO |
WO 2022221425 | Oct 2022 | WO |
WO 2022226057 | Oct 2022 | WO |
WO 2022236054 | Nov 2022 | WO |
WO 2022243303 | Nov 2022 | WO |
WO 2022226372 | Dec 2022 | WO |
WO 2022256503 | Dec 2022 | WO |
WO 2022271820 | Dec 2022 | WO |
WO 2023287765 | Jan 2023 | WO |
WO 2023018799 | Feb 2023 | WO |
WO 2023034489 | Mar 2023 | WO |
WO 2023076345 | May 2023 | WO |
WO 2023086880 | May 2023 | WO |
WO 2023102118 | Jun 2023 | WO |
WO 2023150098 | Aug 2023 | WO |
WO 2023150163 | Aug 2023 | WO |
WO 2023150171 | Aug 2023 | WO |
WO 2023215552 | Nov 2023 | WO |
WO 2023225519 | Nov 2023 | WO |
WO 2023229988 | Nov 2023 | WO |
WO 2023250077 | Dec 2023 | WO |
WO 2024015578 | Jan 2024 | WO |
Entry |
---|
Belaghzal et al., “Hi-C 2.0: An Optimized Hi-C Procedure for High-Resolution Genome-Wide Mapping of Chromosome Conformation,” Methods, Jul. 1, 2017, 123:56-65, 20 pages. |
Belton et al., “Hi-C: A comprehensive technique to capture the conformation of genomes,” Methods, Nov. 2012, 58(3):268-276, 16 pages. |
Bentzen et al., “Large-scale detection of antigen-specific T cells using peptide-MHC-I multimers labeled with DNA barcodes,” Nat Biotechnol., Oct. 2016, 34(10):1037-1045, 12 pages. |
Fan et al., “Illumina Universal Bead Arrays,” Methods in Enzymology, 2006, 410:57-73. |
Hadrup et al., “Parallel detection of antigen-specific T-cell responses by multidimensional encoding of MHC multimers,” Nat. Methods., Jul. 2009, 6(7), 520-526. |
Mamedov et al., “Preparing unbiased T-cell receptor and antibody cDNA libraries for the deep next generation sequencing profiling,” Frontiers in Immunol., Dec. 23, 2013, 4(456):1-10. |
Oksuz et al., “Systematic evaluation of chromosome conformation capture assays,” Nature Methods, Sep. 2021, 18:1046-1055. |
Rohland et al., “Partial uracil-DNA-glycosylase treatment for screening of ancient DNA,” Phil. Trans. R. Soc. B, Jan. 19, 2015, 370(1660): 20130624, 11 pages. |
Su et al., “Restriction enzyme selection dictates detection range sensitivity in chromatin conformation capture-based variant-to-gene mapping approaches,” bioRxiv, Dec. 15, 2020, 22 pages. |
Chen et al., “Large field of view-spatially resolved transcriptomics at nanoscale resolution,” bioRxiv, Jan. 19, 2021, retrieved from URL <https://www.biorxiv.org/node/1751045.abstract>, 37 pages. |
Cho et al., “Seq-Scope: Submicrometer-resolution spatial transcriptomics for single cell and subcellular studies,” bioRxiv, Jan. 27, 2021, retrieved from URL <https://www.biorxiv.org/node/1754517.abstract>, 50 pages. |
Ergin et al., “Proteomic Analysis of PAXgene-Fixed Tissues,” J Proteome Res., 2010, 9(10):5188-96. |
Mathieson et al., “A Critical Evaluation of the PAXgene Tissue Fixation System: Morphology, Immunohistochemistry, Molecular Biology, and Proteomics,” Am J Clin Pathol., Jul. 8, 2016, 146(1):25-40. |
Xia et al., “Spatial transcriptome profiling by MERFISH reveals subcellular RNA compartmentalization and cell cycle-dependent gene expression”, Proceedings of the National Academy of Sciences, Sep. 2019, 116(39):19490-19499. |
U.S. Appl. No. 16/353,937, filed Mar. 14, 2019, Frisen et al. |
U.S. Appl. No. 17/707,189, filed Mar. 29, 2022, Chell et al. |
[No Author Listed], “Chromium Next GEM Single Cell 3′ Reagent Kits v3.1—User Guide,” 10x Genomics, Document No. CG000204, Nov. 2019, 58 pages. |
[No Author Listed], “Chromium Next GEM Single Cell 3′ Reagent Kits v3.1 (Dual Index)—User Guide,” 10x Genomics, Mar. 2021, Document No. CG000315, 61 pages. |
[No Author Listed], “HuSNP Mapping Assay User's Manual,” Affymetrix Part No. 90094 (Affymetrix, Santa Clara, Calif.), GeneChip, 2000, 104 pages. |
[No Author Listed], “Microarray technologies have excellent possibilities in genomics-related researches,” Science Tools From Amersham Pharmacia Biotech, 1998, 3(4): 8 pages (with English Translation). |
[No Author Listed], “Proseek® Multiplex 96×96 User Manual,” Olink Proteomics, Olink Bioscience, Uppsala, Sweden, 2017, 20 pages. |
10xGenomics.com, [online], “Visium Spatial Gene Expression Reagent Kits—Tissue Optimization—User Guide,” Jul. 2020, retrieved on May 25, 2021, retrieved from URL<https://assets.ctfassets.net/an68im79xiti/5UJrN0CH17rEk0UXwd19It/e54d99fb08a8f1500aba503005a04a56/CG000238_VisiumSpatialTissueOptimizationUserGuide_RevD.pdf>, 42 pages. |
10xGenomics.com, [online], “Visium Spatial Gene Expression Reagent Kits—Tissue Optimization,” Nov. 2019, retrieved on Jan. 25, 2022, retrieved from URL<https://assets.ctfassets.net/an68im79xiti/4q03w6959AJFxffSw51ee9/6a2ac61cf6388a72564eeb96bc294967/CG000238_VisiumSpatialTissueOptimizationUserGuide_Rev_A.pdf>, 46 pages. |
10xGenomics.com, [online], “Visium Spatial Gene Expression Reagent Kits—Tissue Optimization,” Oct. 2020, retrieved on Dec. 28, 2021, retrieved from URL<https://assets.ctfassets.net/an68im79xiti/5UJrN0CH17rEk0UXwd19It/e54d99fb08a8f1500aba503005a04a56/CG000238_VisiumSpatialTissueOptimizationUserGuide_RevD.pdf>, 43 pages. |
10xGenomics.com, [online], “Visium Spatial Gene Expression Reagent Kits—User Guide,” Jun. 2020, retrieved on May 25, 2021, retrieved from URL<https://assets.ctfassets.net/an68im79xiti/3GGIfH3RWpd1bFVha1pexR/8baa08d9007157592b65b2cdc7130990/CG000239_VisiumSpatialGeneExpression_UserGuide_RevD.pdf>, 69 pages. |
10xGenomics.com, [online], “Visium Spatial Gene Expression Reagent Kits—User Guide,” Oct. 2020, retrieved on Dec. 28, 2021, retrieved from URL<https://assets.ctfassets.net/an68im79xiti/3GGIfH3RWpd1bFVha1pexR/8baa08d9007157592b65b2cdc7130990/CG000239_VisiumSpatialGeneExpression_UserGuide_RevD.pdf>, 70 pages. |
Adessi et al., “Solid phase DNA amplification: characterisation of primer attachment and amplification mechanisms,” Nucl. Acids Res., 2000, 28(20):E87, 8 pages. |
Adiconis et al., “Comparative analysis of RNA sequencing methods for degraded or low-input samples,” Nat Methods, Jul. 2013, 10(7):623-9. |
Affymetrix, “GeneChip Human Genome U133 Set,” retrieved from the Internet: on the World Wide Web at affymetrix.com/support/technical/datasheets/hgu133_datasheet.pdf, retrieved on Feb. 26, 2003, 2 pages. |
Affymetrix, “Human Genome U95Av2,” Internet Citation, retrieved from the internet: on the World Wide Web affymetrix.com, retrieved on Oct. 2, 2002, 1 page. |
Alam, “Proximity Ligation Assay (PLA),” Curr Protoc Immunol., Nov. 2018, 123(1):e58, 8 pages. |
Albretsen et al., “Applications of magnetic beads with covalently attached oligonucleotides in hybridization: Isolation and detection of specific measles virus mRNA from a crude cell lysate,” Anal. Biochem., 1990, 189(1):40-50. |
Allawi et al., “Thermodynamics and NMR of Internal GâT Mismatches in DNA,” Biochemistry, 1996, 36(34):10581-10594. |
Amidzadeh et al., “Assessment of different permeabilization methods of minimizing damage to the adherent cells for detection of intracellular RNA by flow cytometry,” Avicenna J Med Biotechnol., Jan. 2014, 6(1):38-46. |
Andresen et al., “Helicase-dependent amplification: use in OnChip amplification and potential for point-of-care diagnostics,” Expert Rev Mol Diagn., Oct. 2009, 9(7):645-650. |
Aran et al., “xCell: digitally portraying the tissue cellular heterogeneity landscape,” Genome Biol., Nov. 2017, 18(1):220, 14 pages. |
Archer et al., “Selective and flexible depletion of problematic sequences from RNA-seq libraries at the cDNA stage,” BMC Genomics, May 2014, 15(1):401, 9 pages. |
Armani et al, “2D-PCR: a method of mapping DNA in tissue sections,” Lab Chip, 2009, 9(24):3526-34. |
Asp et al., “Spatially Resolved Transcriptomes—Next Generation Tools for Tissue Exploration,” Bioessays, Oct. 2020, 42(10):e1900221, 16 pages. |
Atkinson et al., “An Updated Protocol for High Throughput Plant Tissue Sectioning,” Front Plant Sci, 2017, 8:1721, 8 pages. |
Atkinson, “Overview of Translation: Lecture Manuscript,” U of Texas, 2000, DD, pp. 6.1-6.8. |
Bains et al., “A novel method for nucleic acid sequence determination,” Journal of Theoretical Biology, 1988, 135(3), 303-7. |
Balakrishnan et al., “Flap endonuclease 1,” Annu Rev Biochem., Jun. 2013, 82:119-138. |
Barnes, “PCR amplification of up to 35-kb DNA with high fidelity and high yield from lambda bacteriophage templates,” Proc. Natl. Acad. Sci USA, 1994, 91(6):2216-2220. |
Barnett et al., “ATAC-Me Captures Prolonged DNA Methylation of Dynamic Chromatin Accessibility Loci during Cell Fate Transitions,” Mol Cell., Mar. 2020, 77(6):1350-1364.e6. |
Bartosovic et al., “Single-cell CUT&Tag profiles histone modifications and transcription factors in complex tissues,” Nat Biotechnol., Jul. 2021, 39(7):825-835, Abstract. |
Baugh et al., “Quantitative analysis of mRNA amplification by in vitro transcription,” Nucleic Acids Res., 2001, 29(5):e29, 9 pages. |
Beattie et al., “Advances in genosensor research,” Clin Chem., May 1995, 41(5):700-6. |
Beechem et al., “High-Plex Spatially Resolved RNA and Protein Detection Using Digital Spatial Profiling: A Technology Designed for Immuno-oncology Biomarker Discovery and Translational Research,” Methods Mol Biol, 2020, Chapter 25, 2055:563-583. |
Bell, “A simple way to treat PCR products prior to sequencing using ExoSAP-IT,” Biotechniques, 2008, 44(6):834, 1 page. |
Bentley et al., “Accurate whole human genome sequencing using reversible terminator chemistry,” Nature, 2008, 456(7218):53-59. |
Bergenstråhle et al., “Seamless integration of image and molecular analysis for spatial transcriptomics workflows,” BMC Genomics, Jul. 2020, 21(1):482, 7 pages. |
Berger et al., “Universal bases for hybridization, replication and chain termination,” Nucleic Acid Res., Aug. 2000, 28(15):2911-2914. |
Birney et al., “Identification and analysis of functional elements in 1% of the human genome by the ENCODE pilot project,” Nature, 2007, 447(7146):799-816. |
Blair et al., “Microarray temperature optimization using hybridization kinetics,” Methods Mol Biol., 2009, 529:171-96. |
Blanchard et al., “High-density oligonucleotide arrays,” Biosensors & Bioelectronics, 1996, 11(6-7):687-690. |
Blanco et al., “A practical approach to FRET-based PNA fluorescence in situ hybridization,” Methods, Dec. 2010, 52(4):343-51. |
Blokzijl et al., “Profiling protein expression and interactions: proximity ligation as a tool for personalized medicine,” J Intern. Med., 2010, 268(3):232-245. |
Blow, “Tissue Issues,” Nature, 2007, 448(7156):959-962. |
Bolotin et al., “MiXCR: software for comprehensive adaptive immunity profiling,” Nat Methods., May 2015, 12(5):380-1. |
Boulé et al., “Terminal deoxynucleotidyl transferase indiscriminately incorporates ribonucleotides and deoxyribonucleotides,” J Biol Chem., Aug. 2001, 276(33):31388-93. |
Brandon et al., “Mitochondrial mutations in cancer,” Oncogene, 2006, 25(34):4647-4662. |
Brenner et al., “Gene expression analysis by massively parallel signature sequencing (MPSS) on microbead arrays,” Nat. Biotech., 2000, 18(6):630-634. |
Brenner et al., “In vitro cloning of complex mixtures of DNA on microbeads: physical separation of differentially expressed cDNAs,” Proc. Natl. Acad. Sci. USA, 2000, 97(4):1665-1670. |
Brow, “35—The Cleavase I enzyme for mutation and polymorphism scanning,” PCR Applications Protocols for Functional Genomics, 1999, pp. 537-550. |
Brown et al., “Retroviral integration: structure of the initial covalent product and its precursor, and a role for the viral IN protein,” Proc Natl Acad Sci USA, Apr. 1989, 86(8):2525-9. |
Buenrostro et al., “Transposition of native chromatin for multimodal regulatory analysis and personal epigenomics,” Nat Methods, Dec. 2013, 10(12):1213-1218. |
Bullard et al., “Direct comparison of nick-joining activity of the nucleic acid ligases from bacteriophage T4,” Biochem. J. 2006, 398(1):135-144. |
Burgess, “A space for transcriptomics,” Nature Reviews Genetics, 2016, 17(8):436-7. |
Burgess, “Finding structure in gene expression,” Nature Reviews Genetics, 2018, 19(5):249, 1 page. |
Burgess, “Spatial transcriptomics coming of age,” Nat Rev Genet., Jun. 2019, 20(6):317, 1 page. |
Burton et al., “Coverslip Mounted-Immersion Cycled in Situ RT-PCR for the Localization of mRNA in Tissue Sections,” Biotechniques, 1998, 24(1):92-100. |
Caliari et al., “A practical guide to hydrogels for cell culture,” Nat Methods., Apr. 2016, 13(5):405-14. |
Cha et al., “Specificity, efficiency, and fidelity of PCR,” Genome Res., 1993, 3(3):S18-29. |
Chandra et al., “Cell-free synthesis-based protein microarrays and their applications,” Proteomics, 2009, 5(6):717-30. |
Chatterjee et al., “Mitochondrial DNA mutations in human cancer. Oncogene,” 2006, 25(34):4663-4674. |
Chen et al., “DNA hybridization detection in a microfluidic Channel using two fluorescently labelled nucleic acid probes,” Biosensors and Bioelectronics, 2008, 23(12):1878-1882. |
Chen et al., “Expansion microscopy,” Science, 2015, 347(6221):543-548. |
Chen et al., “Nanoscale imaging of RNA with expansion microscopy,” Nat Methods, Aug. 2016, 13(8):679-84. |
Chen et al., “Parallel single nucleotide polymorphism genotyping by surface invasive cleavage with universal detection,” Anal Chem., Apr. 2005, 77(8):2400-5. |
Chen et al., “RNA imaging. Spatially resolved, highly multiplexed RNA profiling in single cells,” Science, Apr. 2015, 348(6233):aaa6090, 21 pages. |
Chen et al., “Spatial Transcriptomics and In Situ Sequencing to Study Alzheimer's Disease,” Cell, Aug. 2020, 182(4):976-991. |
Chen et al., “μCB-seq: microfluidic cell barcoding and sequencing for high-resolution imaging and sequencing of single cells,” Lab Chip, Nov. 2020, 20(21):3899-3913. |
Chester et al., “Dimethyl sulfoxide-mediated primer Tm reduction: a method for analyzing the role of renaturation temperature in the polymerase chain reaction,” Anal Biochem, Mar. 1993, 209(2):284-90. |
Chrisey et al., “Covalent attachment of synthetic DNA to self-assembled monolayer films,” Nucleic Acids Res., Aug. 1996, 24(15):3031-9. |
Ciaccio et al., “Systems analysis of EGF receptor signaling dynamics with microwestern arrays,” Nat Methods, Feb. 2010, 7(2):148-55. |
Constantine et al., “Use of genechip high-density oligonucleotide arrays for gene expression monitoring,” Life Science News, Amersham Life Science, 1998, pp. 11-14. |
Corces et al., “An improved ATAC-seq protocol reduces background and enables interrogation of frozen tissues,” Nat. Methods, 2017, 14(10):959-962. |
Credle et al., “Multiplexed analysis of fixed tissue RNA using Ligation in situ Hybridization,” Nucleic Acids Research, 2017, 45(14):e128, 9 pages. |
Crosetto et al., “Spatially resolved transcriptomics and beyond,” Nature Review Genetics, 2015, 16(1):57-66. |
Cruz et al., “Methylation in cell-free DNA for early cancer detection,” Ann Oncol., Jun. 2018, 29(6):1351-1353. |
Cujec et al., “Selection of v-Abl tyrosine kinase substrate sequences from randomized peptide and cellular proteomic libraries using mRNA display,” Chemistry and Biology, 2002, 9(2):253-264. |
Czarnik, “Encoding methods for combinatorial chemistry,” Curr Opin Chem Biol., Jun. 1997, 1(1):60-6. |
Dahl et al., “Circle-to-circle amplification for precise and sensitive DNA analysis,” Proc. Natl. Acad. Sci., 2004, 101(13):4548-4553. |
Dalma-Weiszhausz et al., “The affymetrix GeneChip platform: an overview,” Methods Enzymol., 2006, 410:3-28. |
Darmanis et al., “ProteinSeq: High-Performance Proteomic Analyses by Proximity, Ligation and Next Generation Sequencing,” PLos One, 2011, 6(9):e25583, 10 pages. |
Daubendiek et al., “Rolling-Circle RNA Synthesis: Circular Oligonucleotides as Efficient Substrates for T7 RNA Polymerase,” J. Am. Chem. Soc., 1995, 117(29):7818-7819. |
Davies et al., “How best to identify chromosomal interactions: a comparison of approaches,” Nat. Methods, 2017, 14(2):125-134. |
Deamer et al., “Characterization of nucleic acids by nanopore analysis,” Acc Chem Res., Oct. 2002, 35(10):817-25. |
Dean et al., “Comprehensive human genome amplification using multiple displacement amplification,” Proc Natl. Acad. Sci. USA, 2002, 99(8):5261-66. |
Deng et al., “Spatial Epigenome Sequencing at Tissue Scale and Cellular Level,” BioRxiv, Mar. 2021, 40 pages. |
Dressman et al., “Transforming single DNA molecules into fluorescent magnetic particles for detection and enumeration of genetic variations,” Proc. Natl. Acad. Sci. USA, 2003, 100(15):8817-8822. |
Drmanac et al., “CoolMPS™: Advanced massively parallel sequencing using antibodies specific to each natural nucleobase,” BioRxiv, 2020, 19 pages. |
Druley et al., “Quantification of rare allelic variants from pooled genomic DNA,” Nat. Methods, 2009, 6(4):263-65. |
Duncan et al., “Affinity chromatography of a sequence-specific DNA binding protein using Teflon-linked oligonucleotides,” Anal. Biochem., 1988, 169(1):104-108. |
Eberwine, “Amplification of mRNA populations using aRNA generated from immobilized oligo(dT)-T7 primed cDNA,” BioTechniques, 1996, 20(4):584-91. |
Eguiluz et al., “Multitissue array review: a chronological description of tissue array techniques, applications and procedures,” Pathology Research and Practice, 2006, 202(8):561-568. |
Eldridge et al., “An in vitro selection strategy for conferring protease resistance to ligand binding peptides,” Protein Eng Des Sel., 2009, 22(11):691-698. |
Ellington et al., “Antibody-based protein multiplex platforms: technical and operational challenges,” Clin Chem, 2010, 56(2):186-193. |
Eng et al., “Profiling the transcriptome with RNA SPOTs,” Nat Methods., 2017, 14(12):1153-1155. |
Evers et al., “The effect of formaldehyde fixation on RNA: optimization of formaldehyde adduct removal,” J Mol Diagn., May 2011, 13(3):282-8. |
Fire et al., “Rolling replication of short DNA circles,” Proc. Natl. Acad. Sci., 1995, 92(10):4641-4645. |
Flanigon et al., “Multiplex protein detection with DNA readout via mass spectrometry,” N. Biotechnol., 2013, 30(2):153-158. |
Fluidigm, “Equivalence of Imaging Mass Cytometry and Immunofluorescence on FFPE Tissue Sections,” White Paper, 2017, 12 pages. |
Fodor et al., “Light-directed, spatially addressable parallel chemical synthesis,” Science, 1995, 251(4995):767-773. |
Forster et al., “A human gut bacterial genome and culture collection for improved metagenomic analyses,” Nature Biotechnology, 2019, 37(2):186-192. |
Frese et al., “Formylglycine aldehyde Tag—protein engineering through a novel post-translational modification,” ChemBioChem., 2009, 10(3):425-27. |
Fu et al., “Continuous Polony Gels for Tissue Mapping with High Resolution and RNA Capture Efficiency,” bioRxiv, 2021, 20 pages. |
Fu et al., “Counting individual DNA molecules by the stochastic attachment of diverse labels,” PNAS, 2011, 108(22):9026-9031. |
Fu et al., “Repeat subtraction-mediated sequence capture from a complex genome,” Plant J., Jun. 2010, 62(5):898-909. |
Fullwood et al., “Next-generation DNA sequencing of paired-end tags (PET) for transcriptome and genome analyses,” Genome Res., 2009, 19(4):521-532. |
Ganguli et al., “Pixelated spatial gene expression analysis from tissue,” Nat Commun., Jan. 2018, 9(1):202, 9 pages. |
Gansauge et al., “Single-stranded DNA library preparation from highly degraded DNA using T4 DNA ligase,” Nucleic Acids Res., Jun. 2017, 45(10):e79, 10 pages. |
Gao et al., “Q&A: Expansion microscopy,” BMC Biology, 15:50, 9 pages, 2017. |
Gene@arrays[online], BeadArray Technology, available on or before Feb. 14, 2015, via Internet Archive: Wayback Machine URL <https://web.archive.org/web/20150214084616/http://genearrays.com/services/microarrays/illumina/beadarray-technology/>, [retrieved on Jan. 30, 2020], 3 pages. |
Gerard et al., “Excess dNTPs minimize RNA hydrolysis during reverse transcription,” Biotechniques, Nov. 2002, 33(5):984, 986, 988, 990. |
Gill et al., “Nucleic acid isothermal amplification technologies: a review,” Nucleosides Nucleotides Nucleic Acids, Mar. 2008, 27(3):224-43. |
Glass et al., “SIMPLE: a sequential immunoperoxidase labeling and erasing method,” J. Histochem. Cytochem., Oct. 2009, 57(10):899-905. |
Gloor, “Gene targeting in Drosophila,” Methods Mol Biol., 2004, 260:97-114. |
Gnanapragasam, “Unlocking the molecular archive: the emerging use of formalin-fixed paraffin-embedded tissue for biomarker research in urological cancer,” BJU International, 2009, 105(2):274-278. |
Goldkorn et al., “A simple and efficient enzymatic method for covalent attachment of DNA to cellulose. Application for hybridization-restriction analysis and for in vitro synthesis of DNA probes,” Nucleic Acids Res., 1986, 14(22):9171-9191. |
Goryshin et al., “Tn5 in vitro transposition,” J Biol Chem., Mar. 1998, 273(13):7367-74. |
Gracia Villacampa et al., “Genome-wide Spatial Expression Profiling in FFPE Tissues,” bioRxiv, 2020, pp. 38 pages. |
Grokhovsky, “Specificity of DNA cleavage by ultrasound,” Molecular Biology, 2006, 40(2):276-283. |
Gu et al., “Protein tag-mediated conjugation of oligonucleotides to recombinant affinity binders for proximity ligation,” N Biotechnol., 2013, 30(2):144-152. |
Gunderson et al., “Decoding randomly ordered DNA arrays,” Genome Research, 2004, 14(5):870-877. |
Guo et al., “Direct fluorescence analysis of genetic polymorphisms by hybridization with oligonucleotide arrays on glass supports,” Nucleic Acids Res., Dec. 1994, 22(24):5456-65. |
Gupta et al., “Single-cell isoform RNA sequencing characterizes isoforms in thousands of cerebellar cells,” Nature Biotechnol., Oct. 2018, 36:1197-1202. |
Hafner et al., “Identification of microRNAs and other small regulatory RNAs using cDNA library sequencing,” Methods, Jan. 2008, 44(1):3-12. |
Hahnke et al., “Striptease on glass: validation of an improved stripping procedure for in situ microarrays,” J Biotechnol., Jan. 2007, 128(1):1-13. |
Hamaguchi et al., “Direct reverse transcription-PCR on oligo(dT)-immobilized polypropylene microplates after capturing total mRNA from crude cell lysates,” Clin Chem., Nov. 1998, 44(11):2256-63. |
Hanauer et al., “Separation of nanoparticles by gel electrophoresis according to size and shape,” Nano Lett., Sep. 2007, 7(9):2881-5. |
Hardenbol et al., “Highly multiplexed molecular inversion probe genotyping: over 10,000 targeted SNPs genotyped in a single tube assay,” Genome Res., Feb. 2005, 15(2):269-75. |
Hardenbol et al., “Multiplexed genotyping with sequence-tagged molecular inversion probes,” Nature Biotechnol., Jun. 2003, 21(6):673-678. |
Hayes et al., “Electrophoresis of proteins and nucleic acids: I-Theory,” BMJ, Sep. 1989, 299(6703):843-6. |
He et al., “In situ synthesis of protein arrays,” Current Opinion in Biotechnology, 2008, 19(1):4-9. |
He et al., “Printing protein arrays from DNA arrays,” Nature Methods, 2008, 5(2):175-77. |
He, “Cell-free protein synthesis: applications in proteomics and biotechnology,” New Biotechnology, 2008, 25(2-3):126-132. |
Healy, “Nanopore-based single-molecule DNA analysis,” Nanomedicine (Lond), Aug. 2007, 2(4):459-81. |
Hejatko et al., “In situ hybridization technique for mRNA detection in whole mount Arabidopsis samples,” Nature Protocols, 2006, 1(4):1939-1946. |
Hessner et al., “Genotyping of factor V G1691A (Leiden) without the use of PCR by invasive cleavage of oligonucleotide probes,” Clin Chem., Aug. 2000, 46(8 Pt 1):1051-6. |
Hiatt et al., “Parallel, tag-directed assembly of locally derived short sequence reads,” Nature Methods, 2010, 7(2):119-25. |
Ho et al., “Bacteriophage T4 RNA ligase 2 (gp24.1) exemplifies a family of RNA ligases found in all phylogenetic domains,” PNAS, Oct. 2002, 99(20):12709-14. |
Ho et al., “Characterization of an ATP-Dependent DNA Ligase Encoded by Chlorella Virus PBCV-1,” Journal of Virology, Mar. 1997, 71(3):1931-1937. |
Hoffman et al., “Formaldehyde crosslinking: a tool for the study of chromatin complexes,” J Biol Chem., Oct. 2015, 290(44):26404-11. |
Hsuih et al., “Novel, Ligation-Dependent PCR Assay for Detection of Hepatitis C Virus in Serum,” Journal of Clinical Microbiology, Mar. 1996, 34(3):501-507. |
Hu et al., “High reproducibility using sodium hydroxide-stripped long oligonucleotide DNA microarrays,” Biotechniques, Jan. 2005, 38(1):121-4. |
Hughes et al., “Microfluidic Western blotting,” PNAS, Dec. 2012, 109(52):21450-21455. |
Hycultbiotech.com, [online], “Immunohistochemistry, Paraffin” Apr. 2010, retrieved on Apr. 16, 2020, retrieved from URL<https://www.hycultbiotech.com/media/wysiwyg/Protocol_Immunohistochemistry_Paraffin_2.pdf>, 3 pages. |
Ichikawa et al., “In vitro transposition of transposon Tn3,” J Biol. Chem., Nov. 1990, 265(31):18829-32, Abstract. |
Illumina.com [online], “Ribo-Zero® rRNA Removal Kit Reference Guide,” Aug. 2016, retrieved on Apr. 26, 2022, retrieved from URL<https://jp.support.illumina.com/content/dam/illumina-support/documents/documentation/chemistry_documentation/ribosomal-depletion/ribo-zero/ribo-zero-reference-guide-15066012-02.pdf>, 36 pages. |
Jamur et al., “Permeabilization of cell membranes.,” Method Mol. Biol., 2010, 588:63-66. |
Jemt et al., “An automated approach to prepare tissue-derived spatially barcoded RNA-sequencing libraries,” Scientific Reports, 2016, 6:37137, 10 pages. |
Jensen et al., “Zinc fixation preserves flow cytometry scatter and fluorescence parameters and allows simultaneous analysis of DNA content and synthesis, and intracellular and surface epitopes,” Cytometry A., Aug. 2010, 77(8):798-804. |
Jucá et al., “Effect of dimethyl sulfoxide on reverse transcriptase activity,” Braz. J. Med. Biol. Res., Mar. 1995, 28(3):285-90. |
Kalantari et al., “Deparaffinization of formalin-fixed paraffin-embedded tissue blocks using hot water instead of xylene,” Anal Biochem., Aug. 2016, 507:71-3. |
Kap et al., “Histological assessment of PAXgene tissue fixation and stabilization reagents,” PLoS One, 2011, 6:e27704, 10 pages. |
Kapteyn et al., “Incorporation of non-natural nucleotides into template-switching oligonucleotides reduces background and improves cDNA synthesis from very small RNA samples,” BMC Genomics, Jul. 2010, 11:413, 9 pages. |
Karmakar et al., “Organocatalytic removal of formaldehyde adducts from RNA and DNA bases,” Nature Chemistry, Aug. 3, 2015, 7(9):752-758. |
Kaya-Okur et al., “CUT&Tag for efficient epigenomic profiling of small samples and single cells,” Apr. 2019, 10(1):1930, 10 pages. |
Ke et al., “In situ sequencing for RNA analysis in preserved tissue and cells,” Nat Methods., Sep. 2013, Supplementary Materials, 29 pages. |
Kennedy-Darling et al., “Measuring the Formaldehyde Protein-DNA Cross-Link Reversal Rate,” Analytical Chemistry, 2014, 86(12):5678-5681. |
Kent et al., “Polymerase θ is a robust terminal transferase that oscillates between three different mechanisms during end-joining” Elife, Jun. 2016, 5:e13740, 25 pages. |
Kirby et al., “Cryptic plasmids of Mycobacterium avium: Tn552 to the rescue,” Mol Microbiol., Jan. 2002, 43(1):173-86. |
Kleckner et al., “Tn10 and IS10 transposition and chromosome rearrangements: mechanism and regulation in vivo and in vitro,” Curr Top Microbiol Immunol., 1996, 204:49-82. |
Korbel et al., “Paired-end mapping reveals extensive structural variation in the human genome,” Science, 2007, 318(5849):420-426. |
Kozlov et al., “A highly scalable peptide-based assay system for proteomics,” PLoS One, 2012, 7(6):e37441, 10 pages. |
Kozlov et al., “A method for rapid protease substrate evaluation and optimization,” Comb Chem High Throughput Screen, 2006, 9(6):481-87. |
Kristensen et al., “High-Throughput Methods for Detection of Genetic Variation,” BioTechniques, Feb. 2001, 30(2):318-332. |
Krzywkowski et al., “Chimeric padlock and iLock probes for increased efficiency of targeted RNA detection,” RNA, Jan. 2019, 25(1):82-89. |
Krzywkowski et al., “Fidelity of RNA templated end-joining by chlorella virus DNA ligase and a novel iLock assay with improved direct RNA detection accuracy,” Nucleic Acids Research, Oct. 2017, 45(18):e161, 9 pages. |
Kumar et al., “Template-directed oligonucleotide strand ligation, covalent intramolecular DNA circularization and catenation using click chemistry,” J Am Chem Soc., May 2007, 129(21):6859-64. |
Kurz et al., “cDNA—protein fusions: covalent protein—gene conjugates for the in vitro selection of peptides and proteins,” ChemBioChem., 2001, 2(9):666-72. |
Kwok, “High-throughput genotyping assay approaches,” Pharmocogenomics, Feb. 2000, 1(1):95-100. |
Lage et al., “Whole genome analysis of genetic alterations in small DNA samples using hyperbranched strand displacement amplification and array-CGH,” Genome Research, 2003, 13(2):294-307. |
Lahiani et al., “Enabling Histopathological Annotations on Immunofluorescent Images through Virtualization of Hematoxylin and Eosin,” J Pathol Inform., Feb. 2018, 9:1, 8 pages. |
Lampe et al., “A purified mariner transposase is sufficient to mediate transposition in vitro,” EMBO J., Oct. 1996, 15(19):5470-9. |
Landegren et al., “Reading bits of genetic information: methods for single-nucleotide polymorphism analysis,” Genome Res., Aug. 1998, 8(8):769-76. |
Langdale et al., “A rapid method of gene detection using DNA bound to Sephacryl,” Gene, 1985, 36(3):201-210. |
Larman et al., “Sensitive, multiplex and direct quantification of RNA sequences using a modified RASL assay,” Nucleic Acids Research, 2014, 42(14):9146-9157. |
Lee et al., “Fluorescent in situ sequencing (FISSEQ) of RNA for gene expression profiling in intact cells and tissues,” Nature Protocols, 2015, 10(3):442-458. |
Lee et al., “Improving the efficiency of genomic loci capture using oligonucleotide arrays for high throughput resequencing,” BMC Genomics, Dec. 2009, 10:646, 12 pages. |
Leriche et al., “Cleavable linkers in chemical biology,” Bioorganic & Medicinal Chemistry, 2012, 20:571-582. |
Li et al., “A photocleavable fluorescent nucleotide for DNA sequencing and analysis,” Proc. Natl. Acad. Sci., 2003, 100(2):414-419. |
Li et al., “An activity-dependent proximity ligation platform for spatially resolved quantification of active enzymes in single cells,” Nat Commun, Nov. 2017, 8(1):1775, 12 pages. |
Li et al., “RASL-seq for Massively Parallel and Quantitative Analysis of Gene Expression,” Curr Protoc Mol Biol., Apr. 2012, 4(13):1-10. |
Li et al., “Review: a comprehensive summary of a decade development of the recombinase polymerase amplification,” Analyst, Dec. 2018, 144(1):31-67. |
Lin et al., “Highly multiplexed imaging of single cells using a high-throughput cyclic immunofluorescence method,” Nat Commun., Sep. 2015, 6:8390, 7 pages. |
Linnarsson, “Recent advances in DNA sequencing methods—general principles of sample preparation,” Experimental Cell Research, 2010, 316(8):1339-1343. |
Liu et al., “High-Spatial-Resolution Multi-Omics Atlas Sequencing of Mouse Embryos via Deterministic Barcoding in Tissue,” BioRxiv, 2019, 55 pages. |
Lizardi et al., “Mutation detection and single-molecule counting using isothermal rolling-circle amplification,” Nat. Genet., 1998, 19(3):225-232. |
Lou et al., “A review of room temperature storage of biospecimen tissue and nucleic acids for anatomic pathology laboratories and biorepositories,” Clin Biochem., Mar. 2014, 47(4-5):267-73. |
Lovatt et al., “Transcriptome in vivo analysis (TIVA) of spatially defined single cells in live tissue,” Nature Methods, 2013, 11(2):190-196. |
Lund et al., “Assessment of methods for covalent binding of nucleic acids to magnetic beads, Dynabeads, and the characteristics of the bound nucleic acids in hybridization reactions,” Nucleic Acids Res., 1988, 16(22):10861-80. |
Lundberg et al., “High-fidelity amplification using a thermostable DNA polymerase isolated from Pyrococcus furiosus,” Gene, 1991, 108(1):1-6. |
Lundberg et al., “Homogeneous antibody-based proximity extension assays provide sensitive and specific detection of low-abundant proteins in human blood,” Nucleic Acids Res., 2011, 39(15):e102, 8 pages. |
Lundberg et al., “Multiplexed homogeneous proximity ligation assays for high-throughput protein biomarker research in serological material,” Mol Cell Proteomics, 2011, 10(4):M110.004978, 11 pages. |
Lundin et al., “Increased throughput by parallelization of library preparation for massive sequencing,” PLoS One, Apr. 2010, 5(4):e10029, 7 pages. |
Lyamichev et al., “Invader assay for SNP genotyping,” Methods Mol Biol., 2003, 212:229-40. |
Lyamichev et al., “Polymorphism identification and quantitative detection of genomic DNA by invasive cleavage of oligonucleotide probes,” Nat Biotechnol., Mar. 1999, 17(3):292-6. |
Lyck et al., “Immunohistochemical markers for quantitative studies of neurons and glia in human neocortex,” J Histochem Cytochem, 2008, 56(3):201-21. |
Lykidis et al., “Novel zinc-based fixative for high quality DNA, RNA and protein analysis,” Nucleic Acids Res., Jun. 2007, 35(12):e85, 10 pages. |
MacBeath et al., “Printing proteins as microarrays for high-throughput function determination,” Science, Sep. 2000, 289(5485):1760-1763. |
MacIntyre, “Unmasking antigens for immunohistochemistry.,” Br J Biomed Sci., 2001, 58(3):190-6. |
McCloskey et al., “Encoding PCR products with batch-stamps and barcodes,” Biochem. Genet., 2007, 45(11-12):761-767. |
Meers et al., “Improved CUT&RUN chromatin profiling tools,” Elife, Jun. 2019, 8:e46314, 16 pages. |
Merritt et al., “Multiplex digital spatial profiling of proteins and RNA in fixed tissue,” Nat Biotechnol, May 2020, 38(5):586-599. |
Metzker, “Sequencing technologies—the next generation,” Nature Reviews Genetics, 2010, 11(1):31-46. |
Miele et al., “Mapping cis- and trans- chromatin interaction networks using chromosome conformation capture (3C),” Methods Mol Biol., 2009, 464:105-21. |
Miller et al., “Basic concepts of microarrays and potential applications in clinical microbiology,” Clinical Microbiology Reviews, 2009, 22(4):611-633. |
Miller et al., “Chapter 11—Solid and Suspension Microarrays for Microbial Diagnostics,” Methods in Microbiology, 2015, 42:395-431. |
Miner et al., “Molecular barcodes detect redundancy and contamination in hairpin-bisulfite PCR,” Nucleic Acids Res., Sep. 2004, 32(17):e135, 4 pages. |
Mishra et al., “Three-dimensional genome architecture and emerging technologies: looping in disease,” Genome Medicine, 2017, 9(1):87, 14 pages. |
Mitra et al., “Digital genotyping and haplotyping with polymerase colonies,” Proc. Natl. Acad. Sci. USA, May 2003, 100(10):5926-5931. |
Miura et al., “Highly efficient single-stranded DNA ligation technique improves low-input whole-genome bisulfite sequencing by post-bisulfite adaptor tagging,” Nucleic Acids Res., Sep. 2019, 47(15):e85, 10 pages. |
Mizusawa et al., “A bacteriophage lambda vector for cloning with BamHI and Sau3A,” Gene, 1982, 20(3):317-322. |
Morlan et al., “Selective depletion of rRNA enables whole transcriptome profiling of archival fixed tissue,” PLoS One, Aug. 2012, 7(8):e42882, 8 pages. |
Motea et al., “Terminal deoxynucleotidyl transferase: the story of a misguided DNA polymerase,” Biochim Biophys Acta., May 2010, 1804(5):1151-66. |
Mulder et al., “CapTCR-seq: hybrid capture for T-cell receptor repertoire profiling,” Blood Advances, Dec. 2018, 2(23):3506-3514. |
Nadji et al., “Immunohistochemistry of tissue prepared by a molecular-friendly fixation and processing system,” Appl Immunohistochem Mol Morphol., Sep. 2005, 13(3):277-82. |
Nandakumar et al., “How an RNA Ligase Discriminates RNA versus DNA Damage,” Molecular Cell, 2004, 16:211-221. |
Nandakumar et al., “RNA Substrate Specificity and Structure-guided Mutational Analysis of Bacteriophage T4 RNA Ligase 2,” Journal of Biological Chemistry, Jul. 2004, 279(30):31337-31347. |
Ncbi.nlm.nih.gov, [online], “Molecular Inversion Probe Assay,” available on or before Oct. 14, 2014, via Internet Archive: Wayback Machine URL<https://web.archive.org/web/20141014124037/https://www.ncbi.nlm.nih.gov/probe/docs/techmip/>, retrieved on Jun. 16, 2021, retrieved from URL<https://www.ncbi.nlm.nih.gov/probe/docs/techmip/>, 2 pages. |
Ng et al., “Gene identification signature (GIS) analysis for transcriptome characterization and genome annotation,” Nature Methods, 2005, 2(2):105-111. |
Nichols et al., “RNA Ligases,” Curr Protoc Mol Biol., Oct. 2008, 84(1):3.15.1-3.15.4. |
Niedringhaus et al., “Landscape of next-generation sequencing technologies,” Anal Chem., Jun. 2011, 83(12):4327-41. |
Nikiforov et al., “The use of 96-well polystyrene plates for DNA hybridization-based assays: an evaluation of different approaches to oligonucleotide immobilization,” Anal Biochem, May 1995, 227(1):201-9. |
Niklas et al., “Selective permeabilization for the high-throughput measurement of compartmented enzyme activities in mammalian cells,” Anal Biochem, Sep. 2011, 416(2):218-27. |
Nilsson et al., “RNA-templated DNA ligation for transcript analysis,” Nucleic Acids Res., Jan. 2001, 29(2):578-81. |
Nowak et al., “Entering the Postgenome Era,” Science, 1995, 270(5235):368-71. |
Ohtsubo et al., “Bacterial insertion sequences,” Curr Top Microbiol Immunol., 1996, 204:1-26. |
Olivier, “The Invader assay for SNP genotyping,” Mutat. Res., Jun. 2005, 573(1-2):103-110. |
Ozsolak et al., “Digital transcriptome profiling from attomole-level RNA samples,” Genome Res., Apr. 2010, 20(4):519-25. |
Pandey et al., “Inhibition of terminal deoxynucleotidyl transferase by adenine dinucleotides. Unique inhibitory action of Ap5A,” FEBS Lett., Mar. 1987, 213(1):204-8. |
Park et al., “Single cell trapping in larger microwells capable of supporting cell spreading and proliferation,” Microfluid Nanofluid, 2010, 8:263-268. |
Passow et al., “RNAlater and flash freezing storage methods nonrandomly influence observed gene expression in RNAseq experiments,” bioRxiv, Jul. 2018, 28 pages. |
PCT International Preliminary Report on Patentability in International Appln. No. PCT/EP2016/057355, dated Oct. 10, 2017, 7 pages. |
PCT International Preliminary Report on Patentability in International Appln. No. PCT/US2019/048425, dated Mar. 2, 2021, 9 pages. |
PCT International Preliminary Report on Patentability in International Appln. No. PCT/US2019/048434, dated Mar. 2, 2021, 15 pages. |
PCT International Preliminary Report on Patentability in International Appln. No. PCT/US2021/018795, dated Sep. 1, 2022, 10 pages. |
PCT International Preliminary Report on Patentability in International Appln. No. PCT/US2021/018816, dated Sep. 1, 2022, 9 pages. |
PCT International Search Report and Written Opinion in International Appln. No. PCT/US2020/066681, dated Apr. 14, 2021, 17 pages. |
PCT International Search Report and Written Opinion in International Appln. No. PCT/US2021/012659, dated Apr. 16, 2021, 15 pages. |
PCT International Search Report and Written Opinion in International Appln. No. PCT/US2022/028071, dated Aug. 25, 2022, 13 pages. |
Pellestor et al., “The peptide nucleic acids (PNAs), powerful tools for molecular genetics and cytogenetics,” Eur J Hum Genet., Sep. 2004, 12(9):694-700. |
Pemov et al., “DNA analysis with multiplex microarray-enhanced PCR,” Nucl. Acids Res., Jan. 2005, 33(2):e11, 9 pages. |
Penno et al., “Stimulation of reverse transcriptase generated cDNAs with specific indels by template RNA structure: retrotransposon, dNTP balance, RT-reagent usage,” Nucleic Acids Res., Sep. 2017, 45(17):10143-10155. |
Perler et al., “Intervening sequences in an Archaea DNA polymerase gen,” Proc Natl Acad Sci USA, Jun. 1992, 89(12):5577-5581. |
Perocchi et al., “Antisense artifacts in transcriptome microarray experiments are resolved by actinomycin D,” Nucleic Acids Res., 2007, 35(19):e128, 7 pages. |
Petterson et al., “Generations of sequencing technologies,” Genomics, 2009, 93(2):105-111. |
Picelli et al., “Full-length RNA-seq from single cells using Smart-seq2,” Nat Protoc., Jan. 2014, 9(1):171-81. |
Picelli et al., “Tn5 transposase and tagmentation procedures for massively scaled sequencing projects,” Genome Res., Dec. 2014, 24(12):2033-40. |
Pipenburg et al., “DNA detection using recombination proteins,” PLoS Biol., Jul. 2006, 4(7):e204, 7 pages. |
Plasterk, “The Tc1/mariner transposon family,” Curr Top Microbiol Immunol., 1996, 204:125-43. |
Plongthongkum et al., “Advances in the profiling of DNA modifications: cytosine methylation and beyond,” Nature Reviews Genetics, Aug. 2014, 15(10):647-661. |
Polsky-Cynkin et al., “Use of DNA immobilized on plastic and agarose supports to detect DNA by sandwich hybridization,” Clin. Chem., 1985, 31(9):1438-1443. |
Porreca et al., “Polony DNA sequencing,” Curr Protoc Mol Biol., Nov. 2006, Chapter 7, Unit 7.8, pp. 7.8.1-7.8.22. |
U.S. Appl. No. 61/267,363, filed Dec. 7, 2009, 33 pages. |
Qiu et al., “Combination probes with intercalating anchors and proximal fluorophores for DNA and RNA detection,” Nucleic Acids Research, Sep. 2016, 44(17):e138, 12 pages. |
Raab et al., “Human tRNA genes function as chromatin insulators,” EMBO J., Jan. 2012, 31(2):330-50. |
Ranki et al., “Sandwich hybridization as a convenient method for the detection of nucleic acids in crude samples,” Gene, 1983, 21(1-2):77-85. |
Reinartz et al., “Massively parallel signature sequencing (MPSS) as a tool for in-depth quantitative gene expression profiling in all organisms,” Brief Funct Genomic Proteomic, Feb. 2002, 1(1):95-104. |
Reznikoff, “Tn5 as a model for understanding DNA transposition,” Mol Microbiol., Mar. 2003, 47(5):1199-206. |
Ristic et al., “Detection of Protein-Protein Interactions and Posttranslational Modifications Using the Proximity Ligation Assay: Application to the Study of the SUMO Pathway,” Methods Mol. Biol., 2016, 1449:279-90. |
Rodriques et al., “Slide-seq: A scalable technology for measuring genome-wide expression at high spatial resolution,” Science, 2019, 363(6434):1463-1467. |
Ronaghi et al., “A sequencing method based on real-time pyrophosphate,” Science, Jul. 1998, 281(5375):363-365. |
Ronaghi et al., “Real-time DNA sequencing using detection of pyrophosphate release,” Analytical Biochemistry, Nov. 1996, 242(1):84-89. |
Ronaghi, “Pyrosequencing sheds light on DNA sequencing,” Genome Res, Jan. 2001, 11(1):3-11. |
Roy et al., “Assessing long-distance RNA sequence connectivity via RNA-templated DNA-DNA ligation,” eLife, 2015, 4:e03700, 21 pages. |
Salmén et al., “Barcoded solid-phase RNA capture for Spatial Transcriptomics profiling in mammalian tissue sections,” Nature Protocols, Oct. 2018, 13(11):2501-2534. |
Saxonov et al., “10x Genomics, Mastering Biology to Advance Human Health,” PowerPoint, 10x, 2020, 41 pages. |
Schena et al., “Quantitative monitoring of gene expression patterns with a complementary DNA microarray,” Science, Oct. 1995, 270(5235):467-470. |
Schouten et al., “Relative quantification of 40 nucleic acid sequences by multiplex ligation-dependent probe amplification,” Nucleic Acids Res., Jun. 2002, 30(12):e57, 13 pages. |
Schweitzer et al., “Multiplexed protein profiling on microarrays by rolling-circle amplification,” Nature Biotechnology, Apr. 2002, 20(4):359-365. |
Schwers et al., “A high-sensitivity, medium-density, and target amplification-free planar waveguide microarray system for gene expression analysis of formalin-fixed and paraffin-embedded tissue,” Clin. Chem., Nov. 2009, 55(11):1995-2003. |
Shalon et al., “A DNA microarray system for analyzing complex DNA samples using two-color fluorescent probe hybridization,” Genome Res., Jul. 1996, 6(7):639-45. |
Shelbourne et al., “Fast copper-free click DNA ligation by the ring-strain promoted alkyne-azide cycloaddition reaction,” Chem. Commun., 2011, 47(22):6257-6259. |
Shendure et al., “Accurate multiplex polony sequencing of an evolved bacterial genome,” Science, 2005, 309(5741):1728-1732. |
Simonis et al., “Nuclear organization of active and inactive chromatin domains uncovered by chromosome conformation capture-on-chip (4C),” Nat Genet., Nov. 2006, 38(11):1348-54. |
Singh et al., “High-throughput targeted long-read single cell sequencing reveals the clonal and transcriptional landscape of lymphocytes,” Nat Commun., Jul. 2019, 10(1):3120, 13 pages. |
Skene et al., “An efficient targeted nuclease strategy for high-resolution mapping of DNA binding sites,” Elife, Jan. 2017, 6:e21856, 35 pages. |
Slomovic et al., “Addition of poly(A) and poly(A)-rich tails during RNA degradation in the cytoplasm of human cells,” Proc Natl Acad Sci USA, Apr. 2010, 107(16):7407-12. |
Sountoulidis et al., “SCRINSHOT, a spatial method for single-cell resolution mapping of cell states in tissue sections,” PLoS Biol., Nov. 2020, 18(11):e3000675, 32 pages. |
Spiess et al., “A highly efficient method for long-chain cDNA synthesis using trehalose and betaine,” Anal. Biochem., Feb. 2002, 301(2):168-74. |
Spitale et al., “Structural imprints in vivo decode RNA regulatory mechanisms,” Nature, 2015, 519(7544):486-90. |
Stahl et al., “Visualization and analysis of gene expression in tissue sections by spatial transcriptomics,” Science, Jul. 2016, 353(6294):78-82. |
Stahl et al., “Visualization and analysis of gene expression in tissue sections by spatial transcriptomics,” Supplementary Materials, Science, Jul. 2016, 353(6294):78-82, 41 pages. |
Stimpson et al., “Real-time detection of DNA hybridization and melting on oligonucleotide arrays by using optical wave guides,” Proc Natl Acad Sci USA, Jul. 1995, 92(14):6379-83. |
Stoddart et al., “Single-nucleotide discrimination in immobilized DNA oligonucleotides with a biological nanopore,” PNAS USA., May 2009, 106(19):7702-7707. |
Strell et al., “Placing RNA in context and space—methods for spatially resolved transcriptomics,” The FEBS Journal, 2019, 286(8):1468-1481. |
Stroh et al., “Quantum dots spectrally distinguish multiple species within the tumor milieu in vivo,” Nat Med., Jun. 2005, 11(6):678-82. |
Sutherland et al., “Utility of formaldehyde cross-linking and mass spectrometry in the study of protein-protein interactions,” J. Mass Spectrom., Jun. 2008, 43(6):699-715. |
Taylor et al., “Mitochondrial DNA mutations in human disease,” Nature Reviews Genetics, May 2005, 6(5):389-402. |
Tentori et al., “Detection of Isoforms Differing by a Single Charge Unit in Individual Cells,” Chem. Int. Ed., 2016, 55(40):12431-5. |
Tian et al., “Antigen peptide-based immunosensors for rapid detection of antibodies and antigens,” Anal Chem, 2009, 81(13):5218-5225. |
Tijssen et al., “Overview of principles of hybridization and the strategy of nucleic acid assays” in Techniques in Biochemistry and Molecular Biology—Hybridization with Nucleic Acid Probes, 1993, 24(Chapter 2), 65 pages. |
Tolbert et al., “New methods for proteomic research: preparation of proteins with N-terminal cysteines for labeling and conjugation,” Angewandte Chemie International Edition, Jun. 2002, 41(12):2171-4. |
Toubanaki et al., “Dry-reagent disposable biosensor for visual genotyping of single nucleotide polymorphisms by oligonucleotide ligation reaction: application to pharmacogenetic analysis,” Hum Mutat., Aug. 2008, 29(8):1071-8. |
Trejo et al., “Extraction-free whole transcriptome gene expression analysis of FFPE sections and histology-directed subareas of tissue,” PLoS One, Feb. 2019, 14(2):e0212031, 22 pages. |
Tu et al., “TCR sequencing paired with massively parallel 3′ RNA-seq reveals clonotypic T cell signatures,” Nature Immunology, Dec. 2019, 20(12):1692-1699. |
Twyman et al., “Techniques Patents for SNP Genotyping,” Pharmacogenomics, Jan. 2003, 4(1):67-79. |
Ulery et al., “Biomedical Applications of Biodegradable Polymers,” J Polym Sci B Polym Phys., Jun. 2011, 49(12):832-864. |
U.S. Appl. No. 60/416,118 Fan et al., Multiplex Nucleic Acid Analysis Using Archived or Fixed Samples, filed Oct. 3, 2002, 22 pages. |
Van Gelder et al., “Amplified RNA synthesized from limited quantities of heterogeneous cDNA,” Proc. Natl. Acad. Sci. USA, 1990, 87(5):1663-1667. |
Vandenbroucke et al., “Quantification of splice variants using real-time PCR,” Nucleic Acids Research, 2001, 29(13):e68, 7 pages. |
Vandernoot et al., “cDNA normalization by hydroxyapatite chromatography to enrich transcriptome diversity in RNA-seq applications,” Biotechniques, Dec. 2012, 53(6):373-80. |
Vasiliskov et al., “Fabrication of microarray of gel-immobilized compounds on a chip by copolymerization,” Biotechniques, Sep. 1999, 27(3):592-606. |
Vázquez Bernat et al., “High-Quality Library Preparation for NGS-Based Immunoglobulin Germline Gene Inference and Repertoire Expression Analysis,” Front Immunol., Apr. 2019, 10:660, 12 pages. |
Velculescu et al., “Serial analysis of gene expression,” Science, Oct. 1995, 270(5235):484-7. |
Vickovic et al., “High-definition spatial transcriptomics for in situ tissue profiling,” Nat Methods, Oct. 2019, 16(10):987-990. |
Vickovic et al., “SM-Omics: An automated Platform for High-Throughput Spatial Multi-Omics,” bioRxiv, Oct. 2020, 40 pages. |
Vincent et al., “Helicase-dependent isothermal DNA amplification,” EMBO Rep., Aug. 2004, 5(8):795-800. |
Viollet et al., “T4 RNA ligase 2 truncated active site mutants: improved tools for RNA analysis,” BMC Biotechnol., Jul. 2011, 11:72, 14 pages. |
Vogelstein et al., “Digital PCR,” Proceedings of the National Academy of Sciences, Aug. 1999, 96(16):9236-9241. |
Waichman et al., “Functional immobilization and patterning of proteins by an enzymatic transfer reaction,” Analytical chemistry, 2010, 82(4):1478-85. |
Walker et al., “Strand displacement amplification—an isothermal, in vitro DNA amplification technique,” Nucleic Acids Research, 1992, 20(7):1691-1696. |
Wang et al., “Concentration gradient generation methods based on microfluidic systems,” RSC Adv., 2017, 7:29966-29984. |
Wang et al., “Imaging-based pooled CRISPR screening reveals regulators of IncRNA localization,” Proc Natl Acad Sci USA, May 2019, 116(22):10842-10851. |
Wang et al., “Optimization of Process Conditions for Infected Animal Tissues by Alkaline Hydrolysis Technology,” Procedia Environmental Sciences, 2016, 31:366-374. |
Wang et al., “Tagmentation-based whole-genome bisulfite sequencing,” Nature Protocols, Oct. 2013, 8(10):2022-2032. |
Wang et al., “High-fidelity mRNA amplification for gene profiling,” Nature Biotechnology, Apr. 2000, 18(4):457-459. |
Wang, “RNA amplification for successful gene profiling analysis,” J Transl Med., Jul. 2005, 3:28, 11 pages. |
Weinreich et al., “Evidence that the cis Preference of the Tn5 Transposase is Caused by Nonproductive Multimerization,” Genes and Development, Oct. 1994, 8(19):2363-2374. |
Wiedmann et al., “Ligase chain reaction (LCR)—overview and applications,” PCR Methods Appl., Feb. 1994, 3(4):S51-64. |
Wilson et al., “New transposon delivery plasmids for insertional mutagenesis in Bacillus anthracis,” J Microbiol Methods, Dec. 2007, 71(3):332-5. |
Wohnhaas et al., “DMSO cryopreservation is the method of choice to preserve cells for droplet-based single-cell RNA sequencing,” Scientific Reports, Jul. 2019, 9(1):10699, 14 pages. |
Wolf et al., “Rapid hybridization kinetics of DNA attached to submicron latex particles,” Nucleic Acids Res, 1987, 15(7):2911-2926. |
Wong et al., “Direct Site-Selective Covalent Protein Immobilization Catalyzed by a Phosphopantetheinyl Transferase,” J. Am. Chem Soc., 2008, 130(37):12456-64. |
Worthington et al., “Cloning of random oligonucleotides to create single-insert plasmid libraries,” Anal Biochem, 2001, 294(2):169-175. |
Wu et al., “Detection DNA Point Mutation with Rolling-Circle Amplification Chip,” IEEE, 2010 4th International Conference on Bioinformatics and Biomedical Engineering, Jun. 2010, 4 pages. |
Wu et al., “RollFISH achieves robust quantification of single-molecule RNA biomarkers in paraffin-embedded tumor tissue samples,” Commun Biol., Nov. 2018, 1:209, 8 pages. |
Yasukawa et al., “Effects of organic solvents on the reverse transcription reaction catalyzed by reverse transcriptases from avian myeloblastosis virus and Moloney murine leukemia virus,” Biosci Biotechnol Biochem., 2010, 74(9):1925-30. |
Yeakley et al., “A trichostatin A expression signature identified by TempO-Seq targeted whole transcriptome profiling,” PLoS One, May 2017, 12(5):e0178302, 22 pages. |
Yeakley et al., “Profiling alternative splicing on fiber-optic arrays,” Nature biotechnology, 2002, 20:353-358. |
Yershov et al., “DNA analysis and diagnostics on oligonucleotide microchips,” Proc. Natl. Acad. Sci. USA, May 1996, 93(10):4913-4918. |
Yin et al., “Genetically encoded short peptide tag for versatile protein labeling by Sfp phosphopantetheinyl transferase,” PNAS, 2005, 102(44):15815-20. |
Zhang et al., “Archaeal RNA ligase from Thermoccocus kodakarensis for template dependent ligation,” RNA Biol., Jan. 2017, 14(1):36-44. |
Zhang et al., “Assembling DNA through Affinity Binding to Achieve Ultrasensitive Protein Detection,” Angew Chem Int Ed Engl., 2013, 52(41):10698-705. |
Zhang et al., “Binding-induced DNA assembly and its application to yoctomole detection of proteins,” Anal Chem, 2012, 84(2):877-884. |
Zhang et al., “Genome-wide open chromatin regions and their effects on the regulation of silk protein genes in Bombyx mori,” Sci Rep., Oct. 2017, 7(1):12919, 9 pages. |
Zhang et al., “Multiplex ligation-dependent probe amplification (MLPA) for ultrasensitive multiplexed microRNA detection using ribonucleotide-modified DNA probes†,” Chem. Commun., 2013, 49:10013-10015. |
Zhao et al., “Isothermal Amplification of Nucleic Acids,” Chemical Reviews, Nov. 2015, 115(22):12491-12545. |
Zheng et al., “Origins of human mitochondrial point mutations as DNA polymerase gamma-mediated errors,” Mutat. Res., 2006, 599(1-2):11-20. |
Zhou et al., “Genetically encoded short peptide tags for orthogonal protein labeling by Sfp and AcpS phosphopantetheinyl transferases,” ACS Chemical Biol., 2007, 2(5):337-346. |
Zhu et al., “Reverse transcriptase template switching: a SMART approach for full-length cDNA library construction,” Biotechniques, Apr. 2001, 30(4):892-897. |
U.S. Appl. No. 63/033,348, filed Jun. 2, 2020, Bent. |
Arslan et al., “Engineering of a superhelicase through conformational control (Supplementary Materials),” Science, Apr. 17, 2015, 348(6232):344-347, 18 pages. |
Arslan et al., “Engineering of a superhelicase through conformational control,” Science, Apr. 17, 2015, 348(6232):344-347. |
Baner et al., “Signal amplification of padlock probes by rolling circle replication,” Nucleic Acids Res., 1998, 26(22):5073-5078. |
Borm et al., “High throughput Human embryo spatial transcriptome mapping by surface transfer of tissue RNA,” Abstracts Selected Talks, Single Cell Genomics mtg, (SCG2019), 2019, 1 pages (Abstract Only). |
Chen et al., “Efficient in situ barcode sequencing using padlock probe-based BaristaSeq,” Nucleic Acids Res., 2018, 46(4): e22, 11 pages. |
Codeluppi et al., “Spatial organization of the somatosensory cortex revealed by osmFISH,” Nature Methods, Nov. 2018, 15:932-935. |
Dean et al., “Rapid Amplification Of Plasmid And Phage DNA Using Phi29 DNA Polymerase And Multiply-Primed Rolling Circle Amplification,” Genome Research, Jun. 2001, 11:1095-1099. |
Eng et al., “Transcriptome-scale super-resolved imaging in tissues by RNA seqFISH+,” Nature, Apr. 2019, 568(7751):235-239, 37 pages. |
Faruqi et al., “High-throughput genotyping of single nucleotide polymorphisms with rolling circle amplification,” BMC Genomics, Aug. 2001, 2:4, 10 pages. |
Gao et al., “A highly homogeneous expansion microscopy polymer composed of tetrahedron-like monomers,” bioRxiv, Oct. 22, 2019, 23 pages (Preprint). |
Gilar et al., “Study of phosphorothioate-modified oligonucleotide resistance to 3′-exonuclease using capillary electrophoresis,” J Chromatogr B Biomed Sci Appl., Aug. 28, 1998, 714(1):13-20. |
Goh et al., “Highly Specific Multiplexed RNA Imaging In Tissues With Split-FISH,” Nat Methods, Jun. 15, 2020, 17(7):689-693, 21 pages. |
Goransson et al., “A single molecule array for digital targeted molecular analyses,” Nucleic Acids Res., Nov. 25, 2009, 37(1):e7, 9 pages. |
Li et al., “A new GSH-responsive prodrug of 5-aminolevulinic acid for photodiagnosis and photodynamic therapy of tumors,” European Journal of Medicinal Chemistry, Nov. 2019, 181:111583, 9 pages. |
Liu et al., “High-Spatial-Resolution Multi-Omnics Sequencing via Deterministic Barcoding in Tissue,” Cell, Nov. 13, 2020, 183(6):1665-1681, 36 pages. |
Liu et al., “Spatial transcriptome sequencing of FFPE tissues at cellular level,” bioRxiv 788992, Oct. 14, 2020, 39 pages. |
Mignardi et al., “Oligonucleotide gap-fill ligation for mutation detection and sequencing in situ,” Nucleic Acids Research, Aug. 3, 2015, 43(22):e151, 12 pages. |
Mohsen et al., “The Discovery of Rolling Circle Amplification and Rolling Circle Transcription,” Acc Chem Res., Nov. 15, 2016, 49(11):2540-2550, 25 pages. |
Nallur et al., “Signal amplification by rolling circle amplification on DNA microarrays,” Nucleic Acids Res., Dec. 1, 2001, 29(23):e118, 9 pages. |
Raj et al., “Imaging individual mRNA molecules using multiple singly labeled probes,” Nature Methods, Oct. 2008, 5(10):877-879, 9 pages. |
Schweitzer et al., “Immunoassays with rolling circle DNA amplification: A versatile platform for ultrasensitive antigen detection,” Proc. Natl Acad. Sci. USA, May 22, 2000, 97:10113-119. |
Takei et al., “Integrated Spatial Genomics Reveals Global Architecture Of Single Nuclei,” Nature, Jan. 27, 2021, 590(7845):344-350, 53 pages. |
Bibikova et al., “Quantitative gene expression profiling in formalin-fixed paraffin-embedded tissues using universal bead arrays,” The American Journal of Pathology, Nov. 1, 2004, 165(5):1799-1807. |
Chen et al. “Arrayed profiling of multiple glycans on whole living cell surfaces.” Analytical chemistry, Oct. 15, 2013, 85(22):11153-11158. |
Choi et al., “Multiplexed detection of mRNA using porosity-tuned hydrogel microparticles,” Analytical chemistry, Sep. 28, 2012, 84(21):9370-9378. |
Fan et al., “A versatile assay for high-throughput gene expression profiling on universal array matrices,” Genome Research, May 1, 2004, 14(5):878-885. |
Goldmeyer et al., “Development of a novel one-tube isothermal reverse transcription thermophilic helicase-dependent amplification platform for rapid RNA detection,” Journal of Molecular Diagnostics, American Society for Investigative Pathology and the Association for Molecular Pathology, Nov. 1, 2007, 9(5):639-644. |
Sun et al., “Statistical Analysis of Spatial Expression Pattern for Spatially Resolved Transcriptomic Studies,” Nature Methods, Jan. 27, 2020, 17(2): 193-200. |
Svensson et al., “SpatialDE: identification of spatially variable genes,” Nature Methods, May 2018, 15:343-346, 15 pages. |
Appella, “Non-natural nucleic acids for synthetic biology,” Current Opinion in Chemical Biology, Dec. 2009, 13(5-6): 687-696. |
Bunt et al., “FRET from single to multiplexed signaling events,” Biophys Rev. Apr. 2017, 9(2): 119-129. |
Grünweller et al., “Locked Nucleic Acid Oligonucleotides,” BioDrugs, Jul. 2007, 21(4): 235-243. |
Gu et al., “Multiplex single-molecule interaction profiling of DNA-barcoded proteins,” Nature, Sep. 21, 2014, 515:554-557. |
Ma et al., “Isothermal amplification method for next-generation sequencing,” PNAS, Aug. 12, 2013, 110(35):14320-14323. |
Marx, “Method of the Year: spatially resolved transcriptomics,” Nature Methods, 2021, 18(1):9-14. |
Orenstein et al., “γPNA FRET Pair Miniprobes for Quantitative Fluorescent In Situ Hybridization to Telomeric DNA in Cells and Tissue,” Molecules, Dec. 2, 2017, 22(12):2117, 15 pages. |
Zahra et al., “Assessment of Different Permeabilization Methods of Minimizing Damage to the Adherent Cells for Detection of Intracellular RNA by Flow Cytometry,” Avicenna Journal of Medical Biotechnology, Jan. 1, 2014, 6(1):38-46. |
Howell et al., “iFRET: An Improved Fluorescence System for DNA-Melting Analysis,” Genome Research, 2002, 12:1401-1407. |
Nam et al., “Nanoparticle-Based Bio-Bar Codes for the Ultrasensitive Detection of Proteins,” Science, Sep. 26, 2003, 301(5641):1884-1886. |
Redmond et al., “Single-cell TCRseq: paired recovery of entire T-cell alpha and beta chain transcripts in T-cell receptors from single-cell RNAseq,” Genome Med, 2016, 8:80, 12 pages. |
Hobro et al, “An evaluation of fixation methods: Spatial and compositional cellular changes observed by Raman imaging,” Vibrational Spectroscopy, Jul. 2017, 91:31-45. |
Landegren et al., “A Ligase-Mediated Gene Detection Technique,” Science, 1988, 241(4869):1077-1080. |
Schmidl et al., “ChIPmentation: fast, robust, low-input ChIP-seq for histones and transcription factors,” Nature Methods, Oct. 2015, 12:963-965. |
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63068146 | Aug 2020 | US |