Spatial and cellular mapping of biomolecules in situ by high-throughput sequencing

• US 10,655,173 B2
• Filed: 10/17/2014
• Issued: 05/19/2020
• Est. Priority Date: 10/18/2013
• Status: Active Grant

• Current Assignee: Massachusetts Institute of Technology, The Broad Institute, Inc.
• Original Assignee: The Broad Institute, Inc.
• Inventors: Zhang, Feng, Weinstein, Joshua Asher, Regev, Aviv
• Primary Examiner(s): Dines, Karla A
• Application Number:
  US15/030,154
• Publication Number:
  US 20160265046A1
• Time in Patent Office: 2,041 Days
• Field of Search: 506 9
• CPC Class Codes: C12Q 1/6846 Common amplification features
• CPC Class Codes: C12Q 1/6874 involving nucleic acid arrays, e.g. sequencing by hybridisation
• CPC Class Codes: C12Q 2525/161 incorporating target specific and non-target specific sites
• CPC Class Codes: C12Q 2533/107 Probe or oligonucleotide ligation
• CPC Class Codes: C12Q 2543/101 in situ amplification
• CPC Class Codes: C12Q 2563/179 the label being a nucleic acid

Abstract

The present invention relates to molecular microscopy or volumetric imaging by proximal unique molecular identifiers (“UID”) reaction (“VIPUR”) microscopy methods to record the cellular co-localization and/or spatial distributions of arbitrary nucleic acid sequences, or other biomolecules tagged with nucleic sequences. The method involves one or both of two DNA sequence-components such as an α-UID, which may identify the targeted sequences-of-interest themselves and/or spatial beacons relative to which their distances are measured, and a- β-UID, which labels α-UID association events.

Claims

1. A method for single cell mapping comprising

(a) labeling template nucleic acid sequences in fixed cells with unique molecular identifiers (UID'"'"'s), wherein the labeling comprises adding at least one α

-UID to each template nucleic acid, thereby generating uniquely labeled template nucleic acids;

(b) amplifying the labeled template nucleic acids using in situ amplification the amplification reaction also further concatenating individual amplified labeled template nucleic acids together such that each concatenation event results in incorporation of at least one unique β

-UID, wherein the frequency of concatenation events between amplified labeled template nucleic acids is a function of a proximity of each individual labeled template nucleic acid to another labeled template nucleic acid; and

(c) generating a spatially resolved physical mapping of each cell of the fixed cells by isolation and sequencing of the uniquely labeled concatenation events from (b).

2. The method of claim 1, wherein the nucleic acid is DNA.

3. The method of claim 2, wherein the DNA is complementary DNA (cDNA).

4. The method of claim 1, wherein the nucleic acid is RNA.

5. The method of claim 1, wherein the cell is fixed and permeabilized.

6. The method of claim 1, wherein the at least one UID is a completely random or computationally designed 10 to 50 mer between a gene-specific and adapter region of a primer used for reverse transcription.

7. The method of claim 1, further comprising cross-linking amplification products of steps (b) and/or (c).

8. The method of claim 7, wherein the cross-linking further comprises an additional UID-labeling step.

9. The method of claim 7, wherein the cross-linking comprises overlap extension polymerase chain reaction.

10. The method of claim 7, wherein the cross-linking comprises associating multiple amplification products with ligase or capture beads.

11. The method of claim 1, wherein the amplification comprises thermocycling or isothermal amplification.

12. The method of claim 1, wherein the amplification occurs in solution, in a hydrogel, or on a solid support.