Spatially distinguished, multiplex nucleic acid analysis of biological specimens

Patent metadata

Field	Value
Publication number	US-11613773-B2
Application number	US-202217834474-A
Country	US
Kind code	B2
Filing date	Jun 7, 2022
Priority date	Apr 10, 2015
Publication date	Mar 28, 2023
Grant date	Mar 28, 2023

Abstract

A method for spatially tagging nucleic acids of a biological specimen, including steps of (a) providing a solid support comprising different nucleic acid probes that are randomly located on the solid support, wherein the different nucleic acid probes each includes a barcode sequence that differs from the barcode sequence of other randomly located probes on the solid support; (b) performing a nucleic acid detection reaction on the solid support to locate the barcode sequences on the solid support; (c) contacting a biological specimen with the solid support that has the randomly located probes; (d) hybridizing the randomly located probes to target nucleic acids from portions of the biological specimen; and (e) modifying the randomly located probes that are hybridized to the target nucleic acids, thereby producing modified probes that include the barcode sequences and a target specific modification, thereby spatially tagging the nucleic acids of the biological specimen.

First claim

The invention claimed is:

1. A composition comprising:
a solid support comprising nucleic acid probes, wherein a nucleic acid probe comprises:
i) a spatial tag sequence that differs from the spatial tag sequence of all other nucleic acid probes on the solid support, and
ii) a capture sequence that hybridizes to a nucleic acid sequence from a biological specimen, wherein the nucleic acid probes are randomly located on the solid support and wherein the spatial tag sequence and location of the nucleic acid probe on the solid support is determined prior to contacting the biological specimen with the solid support.

2. The composition of claim 1 , wherein the solid support comprises a pattern of discrete features.

3. The composition of claim 2 , wherein a feature in the pattern of discrete features is selected from the group consisting of: pits, wells, channels, ridges, raised regions, pegs, posts, and beads.

4. The composition of claim 2 , wherein the features in the pattern of discrete features on the solid support have an average pitch of less than 1 micron.

5. The composition of claim 1 , wherein the solid support further comprises fiducial markers.

6. The composition of claim 1 , wherein the solid support is a flowcell.

7. The composition of claim 1 , wherein the solid support is a slide.

8. The composition of claim 1 , wherein the solid support comprises a gel coating, and wherein the nucleic acid probes are attached to the gel coating.

9. The composition of claim 1 , wherein the nucleic acid probe further comprises a unique molecule identifier sequence.

10. The composition of claim 1 , wherein the nucleic acid probe further comprises a functional sequence, wherein the functional sequence is a universal primer binding site.

11. The composition of claim 1 , wherein the capture sequence that hybridizes to a nucleic acid sequence is a common capture sequence for each nucleic acid probe.

12. The composition of claim 11 , wherein the common capture sequence is a poly(T) sequence.

13. The composition of claim 1 , further comprising the biological specimen, wherein the biological specimen is on the surface of the solid support.

14. The composition of claim 13 , wherein the biological specimen is permeabilized.

15. The composition of claim 14 , comprising the nucleic acid sequence from the biological specimen hybridized to the capture sequence.

16. The composition of claim 15 , further comprising a complementary copy of the nucleic acid sequence from the biological specimen that is hybridized to the capture sequence.

17. The composition of claim 16 , further comprising a polymerase.

18. The composition of claim 17 , wherein the polymerase is a DNA polymerase, an RNA polymerase, or a reverse transcriptase.

19. The composition of claim 1 , wherein the biological specimen is a mixture of cells.

20. The composition of claim 1 , wherein the biological specimen is a tissue section.

21. The composition of claim 20 , wherein the tissue section is a fresh-frozen tissue section.

22. The composition of claim 20 , wherein the tissue section is a fixed tissue section.

23. The composition of claim 22 , wherein the fixed tissue section is a formalin-fixed paraffin-embedded tissue section that was previously deparaffinized.

24. The composition of claim 1 , wherein the biological specimen is from a human.

25. The composition of claim 1 , wherein the biological specimen is from a mammal, a plant, an algae, an insect, a nematode, a fish, a reptile, or a fungi.

26. The composition of claim 1 , wherein the nucleic acid sequence from the biological specimen is a RNA.

27. The composition of claim 26 , wherein the RNA is an mRNA.

28. The composition of claim 1 , wherein the nucleic acid sequence from the biological specimen is genomic DNA.

Assignees

• Spatial Transcriptomics Ab
• Illumina Inc

Inventors

• Frisen Jonas
• Stahl Patrik
• Lundeberg Joakim
• Cann Gordon M
• Bazargan Leila
• Aravanis Alex

Classifications

• C12Q1/6874

  involving nucleic acid arrays, e.g. sequencing by hybridisation · CPC title

• C12N15/1065

  Preparation or screening of tagged libraries, e.g. tagged microorganisms by STM-mutagenesis, tagged polynucleotides, gene tags · CPC title

• C12Q1/6841 Primary

  In situ hybridisation · CPC title

• C12Q1/6834 Primary

  Enzymatic or biochemical coupling of nucleic acids to a solid phase · CPC title

• C12Q1/6876

  Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes · CPC title