METHODS AND SYSTEMS FOR DETERMINING SPATIAL PATTERNS OF BIOLOGICAL TARGETS IN A SAMPLE

• US 20180201980A1
• Filed: 12/04/2017
• Published: 07/19/2018
• Est. Priority Date: 06/25/2013
• Status: Abandoned Application

• Current Assignee: Prognosys Biosciences Inc.
• Original Assignee: Prognosys Biosciences Inc.
• Inventors: Chee, Mark S., Routenberg, David A.
• Application Number:
  US15/831,158
• Publication Number:
  US 20180201980A1
• Time in Patent Office: Days
• CPC Class Codes: B01L 3/5027 by integrated microfluidic structures, i.e. dimensions of channels and chambers are such that surface tension forces are important, e.g. lab-on-a-chip B01L3/5023 takes precedence; micromixers B01F33/30; microreactors for synthesis B01J19/0093; microcapillary devices in general B81B1/00
• CPC Class Codes: C12Q 1/6809 Methods for determination or identification of nucleic acids involving differential detection
• CPC Class Codes: C12Q 1/6841 In situ hybridisation
• CPC Class Codes: C12Q 1/6853 using modified primers or templates
• CPC Class Codes: C12Q 2543/101 in situ amplification
• CPC Class Codes: C12Q 2563/179 the label being a nucleic acid
• CPC Class Codes: C12Q 2565/513 characterised by the pattern of the arrayed oligonucleotides
• CPC Class Codes: C12Q 2565/514 characterised by the use of the arrayed oligonucleotides as identifier tags, e.g. universal addressable array, anti-tag or tag complement array
• CPC Class Codes: C12Q 2565/629 being a microfluidic device

Abstract

The present disclosure provides methods and assay systems for use in spatially encoded biological assays, including assays to determine a spatial pattern of abundance, expression, and/or activity of one or more biological targets across multiple sites in a sample. In particular, the biological targets comprise proteins, and the methods and assay systems do not depend on imaging techniques for the spatial information of the targets. The present disclosure provides methods and assay systems capable of high levels of multiplexing where reagents are provided to a biological sample in order to address tag the sites to which reagents are delivered; instrumentation capable of controlled delivery of reagents; and a decoding scheme providing a readout that is digital in nature.

Claims

1-56. -56. (canceled)

57. A method of determining a spatial pattern of abundance, expression, and/or activity of one or more biological targets across multiple sites in a sample, comprising:

delivering a probe for each of one or more biological targets to multiple sites in a sample, wherein each probe comprises a target-binding moiety capable of binding to the probe'"'"'s corresponding biological target;

allowing each probe to bind to its corresponding biological target in the sample;

delivering at least one adaptor to the multiple sites in the sample, wherein the at least one adaptor specifically binds to the probe and comprises an address tag that identifies each of the multiple sites to which the at least one adaptor is delivered, wherein the probe and/or the adaptor comprises an identity tag that identifies the probe'"'"'s and/or adaptor'"'"'s corresponding biological target or target-binding moiety;

analyzing the at least one adaptor and the probe bound to the one or more biological targets, the analysis comprising;

(1) determining abundance, expression, and/or activity of each of the one or more biological targets by assessing the amount of at least one adaptor bound to the probe bound to the biological target; and

(2) determining the identities of the identity tag, and the address tag of the at least one adaptor; and

determining a spatial pattern of abundance and/or activity of the one or more biological targets across the multiple sites in the sample based on the analysis,

wherein the method does not depend on an imaging technique for determining spatial information of the one or more biological targets in the sample.

61. The method of claim 57, wherein at least two adaptors are delivered to each of the multiple sites in the sample, wherein the at least two adaptors each specifically binds to one probe that specifically binds to the biological target.

62. The method of claim 61, wherein the at least two adaptors are joined.

63. The method of claim 62, wherein the at least two adaptors are ligated by using a portion of the probe sequence as a splint.

64. The method of claim 57, wherein the probe for each of the one or more biological targets comprises an affinity agent for the biological target and an oligonucleotide, and the adaptor comprises an oligonucleotide.

58. A method of determining a spatial pattern of abundance, expression, and/or activity of one or more biological targets across multiple sites in a sample, comprising:

delivering a probe for each of one or more biological targets to multiple sites in a sample, wherein each probe comprises a target-binding moiety capable of binding to the probe'"'"'s corresponding biological target;

allowing each probe to bind to its corresponding biological target in the sample;

delivering at least one adaptor to the multiple sites in the sample, wherein the at least one adaptor specifically binds to the probe and comprises an address tag that identifies each of the multiple sites to which the at least one adaptor is delivered, wherein the probe and/or the adaptor comprises an identity tag that identifies the probe'"'"'s and/or adaptor'"'"'s corresponding biological target or target-binding moiety;

analyzing the at least one adaptor and the probe bound to the one or more biological targets by sequencing, wherein the amount of a sequencing product indicates abundance, expression, and/or activity of each of the one or more biological targets, the sequencing product comprising all or a portion of the address tag sequence and all or a portion of the identity tag sequence; and

determining a spatial pattern of abundance, expression, and/or activity of the one or more biological targets across the multiple sites in the sample based on the analysis,

wherein the method does not depend on an imaging technique for determining spatial information of the one or more biological targets in the sample.

65. The method of claim 58, wherein at least two adaptors are delivered to each of the multiple sites in the sample, wherein the at least two adaptors each specifically binds to one probe that specifically binds to the biological target.

66. The method of claim 65, wherein the at least two adaptors are joined.

67. The method of claim 66, wherein the at least two adaptors are ligated by using a portion of the probe sequence as a splint.

68. The method of claim 58, wherein the probe for each of the one or more biological targets comprises an affinity agent for the biological target and an oligonucleotide, and the adaptor comprises an oligonucleotide.

59. A method of determining a spatial pattern of abundance, expression, and/or activity of one or more biological targets across multiple sites in a sample, comprising:

delivering a probe for each of one or more biological targets to multiple sites in a sample, wherein each probe comprises a target-binding moiety capable of binding to the probe'"'"'s corresponding biological target;

allowing each probe to bind to its corresponding biological target in the sample;

delivering at least one adaptor to the multiple sites in the sample, wherein the at least one adaptor specifically binds to the probe, wherein the probe and/or the adaptor comprises an identity tag that identifies the probe'"'"'s and/or adaptor'"'"'s corresponding biological target or target-binding moiety;

delivering an address tag to each of the multiple sites in the sample, wherein the address tag is to be coupled to the at least one adaptor bound to the probe bound to the biological target and identifies the site to which the address tag is delivered;

analyzing the adaptor/address tag conjugate, the analysis comprising;

(1) determining abundance, expression, and/or activity of each of the one or more biological targets by assessing the amount of the adaptor/address tag conjugate bound to the probe bound to the biological target; and

(2) determining the identities of the identity tag, and the address tag of the adaptor/address tag conjugate; and

determining a spatial pattern of abundance, expression, and/or activity of the one or more biological targets across the multiple sites in the sample based on the analysis,

wherein the method does not depend on an imaging technique for determining spatial information of the one or more biological targets in the sample.

69. The method of claim 59, wherein at least two adaptors are delivered to each of the multiple sites in the sample, wherein the at least two adaptors each specifically binds to one probe that specifically binds to the biological target.

70. The method of claim 69, wherein the at least two adaptors are joined.

71. The method of claim 70, wherein the at least two adaptors are ligated by using a portion of the probe sequence as a splint.

72. The method of claim 59, wherein the probe for each of the one or more biological targets comprises an affinity agent for the biological target and an oligonucleotide, and the adaptor comprises an oligonucleotide.

60. A method of determining a spatial pattern of abundance, expression, and/or activity of one or more biological targets across multiple sites in a sample, comprising:

delivering a probe for each of one or more biological targets to multiple sites in a sample, wherein each probe comprises a target-binding moiety capable of binding to the probe'"'"'s corresponding biological target;

allowing each probe to bind to its corresponding biological target in the sample;

delivering at least one adaptor to the multiple sites in the sample, wherein the at least one adaptor specifically binds to the probe, wherein the probe and/or the adaptor comprises an identity tag that identifies the probe'"'"'s and/or adaptor'"'"'s corresponding biological target or target-binding moiety;

delivering an address tag to each of the multiple sites in the sample, wherein the address tag is to be coupled to the at least one adaptor bound to the probe bound to the biological target and identifies the site to which the address tag is delivered;

analyzing the adaptor/address tag conjugate by sequencing, wherein the amount of a sequencing product indicates abundance, expression, and/or activity of each of the one or more biological targets, the sequencing product comprising all or a portion of the address tag sequence and all or a portion of the identity tag sequence; and

determining a spatial pattern of abundance, expression, and/or activity of the one or more biological targets across the multiple sites in the sample based on the analysis.

73. The method of claim 60, wherein at least two adaptors are delivered to each of the multiple sites in the sample, wherein the at least two adaptors each specifically binds to one probe that specifically binds to the biological target.

74. The method of claim 73, wherein the at least two adaptors are joined.

75. The method of claim 74, wherein the at least two adaptors are ligated by using a portion of the probe sequence as a splint.

76. The method of claim 60, wherein the probe for each of the one or more biological targets comprises an affinity agent for the biological target and an oligonucleotide, and the adaptor comprises an oligonucleotide.