Compositions and methods for nucleic acid sequencing

Patent metadata

Field	Value
Publication number	US-12227799-B2
Application number	US-202117542343-A
Country	US
Kind code	B2
Filing date	Dec 3, 2021
Priority date	Feb 6, 2019
Publication date	Feb 18, 2025
Grant date	Feb 18, 2025

Abstract

Provided herein are methods and compositions for improved sequencing techniques using, for example, polymeric particles and/or three-dimensional structures.

First claim

What is claimed is:

1. A method of sequencing target polynucleotides in a polymer scaffold comprising polynucleotide primers, wherein said polynucleotide primers are covalently attached to the polymer scaffold, the method comprising:
(a) amplifying the target polynucleotides to produce discrete amplicon clusters within the polymer scaffold,
wherein amplifying comprises rolling circle amplification the amplicon clusters are arranged at a plurality of depths; and
(b) sequencing the amplicon clusters, wherein sequencing comprises detecting sequences of signals within the polymer scaffold at a first depth and a second depth.

2. The method of claim 1 , wherein the amplicon clusters have a mean or median separation from one another of about 500-5000 nm.

3. The method of claim 1 , wherein the amplicon clusters have a mean or median diameter of about 100-2000 nm, or about 200-1000 nm.

4. The method of claim 1 , wherein the scaffold polymer comprises water, and optionally wherein the scaffold polymer has a refractive index of about 1.3 when hydrated.

5. The method of claim 1 , wherein the scaffold polymer is a hydrogel.

6. The method of claim 1 , wherein step (a) further comprises contacting the polymer scaffold with one or more reagents for amplifying the target polynucleotides.

7. The method of claim 1 , wherein the sequencing of step (b) comprises (i) extending a sequencing primer to incorporate a detectable label that indicates the identity of a nucleotide in the target polynucleotide, (ii) detecting the detectable label, and (iii) repeating the extending and detecting of steps (i) and (ii).

8. The method of claim 1 , wherein the polymer scaffold is formed by a process comprising:
(a) forming an emulsion of oil droplets in a hydrophilic continuous phase, wherein the hydrophilic continuous phase comprises one or more monomers;
(b) polymerizing the one or more monomers to form the polymer scaffold; and
(c) removing the oil to form a plurality of interconnected pores in the polymer scaffold.

9. The method of claim 1 , wherein the first depth and second depth are separated by 0.1-10 microns.

10. The method of claim 1 , wherein the target polynucleotides are circular.

11. The method of claim 1 , further comprising detecting sequences of signals within the polymer scaffold at a plurality of depths.

12. The method of claim 1 , wherein the polymer scaffold is permeable to a polymerase.

13. The method of claim 1 , wherein the sequencing of step (b) comprises a sequencing-by-synthesis (SBS) process.

14. The method of claim 1 , wherein amplifying the target polynucleotides comprises bridge amplification or an isothermal amplification reaction.

15. The method of claim 1 , wherein the target polynucleotide comprises a primer binding sequence.

16. The method of claim 1 , wherein the polymer scaffold comprises a polymer of one or more of GMA (glicydyl methacrylate), HEMA (hydroxyethylmethacrylate), HEA (hydroxyethylacrylate), HPMA (hydroxypropylmethacrylate), polyacrylamide, poly-N-isopropylacrylamide, poly N-isopropylpolyacrylamide, 2-hydroxyethyl acrylate, polyethylene glycol acrylate, and polyethylene glycol methacrylate, or a copolymer thereof.

17. The method of claim 1 , wherein the polymer scaffold is attached to a glass surface.

18. The method of claim 1 , wherein the polymer scaffold is in a channel of a flow cell.

19. The method of claim 1 , wherein sequencing comprises detecting a series of fluorescent emissions in a first xy plane at the first depth and a series of fluorescent emissions in a second xy plane at the second depth.

20. The method of claim 1 , wherein the first depth and a second depth are separated by greater than 500 nm.

Assignees

• Singular Genomics Systems Inc

Inventors

• Glezer Eli N
• Varnosfaderani Mohammad Vatankhah
• Witters Daan
• Karimkhani Vahid

Classifications

• G01N33/544

  the carrier being organic · CPC title

• G01N33/5308

  for analytes not provided for elsewhere, e.g. nucleic acids, uric acid, worms, mites · CPC title

• C12Q1/6874 Primary

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

• C12Q1/6844

  Nucleic acid amplification reactions · CPC title

• C12Q2565/601

  being a microscope, e.g. atomic force microscopy [AFM] · CPC title