Compositions and methods for nucleic acid sequencing

Patent metadata

Field	Value
Publication number	US-11236387-B2
Application number	US-202017030886-A
Country	US
Kind code	B2
Filing date	Sep 24, 2020
Priority date	Feb 6, 2019
Publication date	Feb 1, 2022
Grant date	Feb 1, 2022

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 amplifying a target polynucleotide, the method comprising:
(a) contacting a composition comprising a plurality of cores with a sample comprising a target polynucleotide, wherein (i) each core of the plurality of cores is surrounded by a shell polymer, (ii) each core is formed by polymerized units of core monomers forming a core polymer, (iii) a core polynucleotide primer is attached to the core polymer within each core, (iv) the shell polymer is formed by polymerized units of shell monomers, and (v) the shell polymer is not attached to a polynucleotide primer;
(b) amplifying the target polynucleotide to produce an amplicon, wherein amplifying comprises extension of the core primer hybridized to the target polynucleotide within each core.

2. The method of claim 1 , further comprising: (c) sequencing the amplicon, wherein sequencing comprises detecting a sequence of signals within each core.

3. The method of claim 1 , wherein the plurality of cores are arranged in a two-dimension array.

4. The method of claim 1 , wherein the plurality of cores are arranged in multiple two-dimensional planes.

5. The method of claim 1 , wherein the core polymer, the shell polymer, or both have a refractive index of about 1.3 when hydrated.

6. The method of claim 1 , wherein step (b) further comprises contacting the plurality of cores with one or more polynucleotide amplification reagents.

7. The method of claim 1 , wherein the sample comprises a plurality of target polynucleotides at a concentration selected such that a majority of each core in the plurality of cores in which the amplification occurs comprise amplicons of only one original target polynucleotide.

8. The method of claim 2 , wherein the sequencing of step (c) 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) Previously presented the extending and detecting of steps (i) and (ii).

9. The method of claim 2 , wherein the detecting of step (c) comprises imaging through each of the multiple two-dimensional planes at a resolution sufficient to distinguish one imaged plane from an adjacent imaged plane.

10. The method of claim 2 , wherein (i) the amplifying of step (b) comprises amplifying a target polynucleotide in two or more cores in the plurality of particles, and (ii) the sequencing of step (c) comprises sequencing an amplicon in two or more cores in the plurality of particles.

11. The method of claim 1 , wherein each of the plurality of cores further comprises a silica, magnetic, or paramagnetic bead.

12. The method of claim 1 , wherein amplifying comprises polymerase chain reaction (PCR), strand displacement amplification (SDA), transcription mediated amplification (TMA), or nucleic acid sequence based amplification (NASBA).

13. The method of claim 1 , wherein amplifying comprises bridge-PCR amplification, recombinase polymerase amplification (RPA), loop-mediated isothermal amplification (LAMP), rolling circle amplification (RCA), strand displacement amplification, or exponential rolling circle amplification (eRCA).

14. The method of claim 1 , wherein each core comprises multiple copies of the core polynucleotide primer.

15. A method of sequencing target polynucleotides, the method comprising:
(a) contacting a plurality of particles with a sample comprising target polynucleotides, wherein each particle comprises a polymer covalently attached to polynucleotide primers;
(b) amplifying the target polynucleotides to produce discrete amplicon clusters, wherein (i) amplifying comprises extension of the primers along the target polynucleotides, (ii) each amplicon cluster originates from amplification of a single target polynucleotide, and (iii) the amplicon clusters are arranged in multiple two-dimensional planes; and
(c) sequencing the amplicon clusters, wherein sequencing comprises detecting sequences of signals from the particles through each of the plurality of two-dimensional planes;

wherein each particle comprises a core surrounded by a shell polymer, wherein:
(a) each core is formed by polymerized units of core monomers forming a core polymer;
(b) a core polynucleotide primer is attached to the core polymer within each core;
(c) a target nucleic acid is capable of hybridizing to the core polynucleotide primer;
(d) the shell polymer is formed by polymerized units of shell monomers; and
(e) the shell polymer is not attached to a polynucleotide primer.

16. The method of claim 15 , wherein the core polymer and shell polymer are permeable to a polymerase.

17. The method of claim 15 , wherein each core further comprises a silica, magnetic, or paramagnetic bead.

18. The method of claim 15 , wherein the plurality of particles comprises water, and optionally wherein the plurality of particles has a refractive index of about 1.3 when hydrated.

19. The method of claim 15 , wherein step (b) further comprises contacting the plurality of particles with one or more polynucleotide amplification reagents.

20. The method of claim 15 , wherein the sequencing of step (c) 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).

21. The method of claim 15 , wherein the detecting comprises imaging through each of the multiple two-dimensional planes at a resolution sufficient to distinguish one imaged plane from an adjacent imaged plane.

22. The method of claim 15 , wherein the imaging comprises confocal microscopy, light sheet fluorescence microscopy, or multi-photon microscopy.

23. The method of claim 15 , wherein the amplicon clusters have a mean or median separation from one another of about 500-5000 nm.

24. The method of claim 15 , wherein the amplicon clusters have a mean or median diameter of about 100-2000 nm, or about 200-1000 nm.

25. The method of claim 15 , wherein the plurality of particles are arranged in a two-dimensional array.

26. The method of claim 15 , wherein the plurality of particles are contained in a channel of a flow cell.

27. The method of claim 15 , wherein the plurality of particles are arranged in multiple two-dimensional planes.

28. The method of claim 15 , wherein amplifying the target polynucleotides comprises an isothermal amplification reaction.

29. The method of claim 15 , wherein amplifying the target polynucleotides comprises bridge amplification.

30. The method of claim 15 , wherein each particle of the plurality of particles comprises a polymer of one or more of GMA (glicydyl methacrylate), HEMA (hydroxyethylmethacrylate), HEA (hydroxyethylacrylate), HPMA (hydroxypropylmethacrylate), or copolymer thereof.

Assignees

• Singular Genomics Systems Inc

Inventors

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

Classifications

• 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

• C12Q1/6869 Primary

  Methods for sequencing · CPC title

• C12Q2533/101

  Primer extension · CPC title