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