Enhancing clustering efficiency and kinetics

Patent metadata

Field	Value
Publication number	US-2023416435-A1
Application number	US-202318330298-A
Country	US
Kind code	A1
Filing date	Jun 6, 2023
Priority date	Jun 7, 2022
Publication date	Dec 28, 2023
Grant date	—

Abstract

A co-polymer includes a plurality of a first monomer including a terminal functional group that is to attach to at least two different primers; a plurality of a second monomer including a second functional group that is different from the terminal functional group, and that is selected from the group consisting of a phenyl group, methoxy propyl, glycosyl, vinyl pyrrolidone, and an imidazole group; and a plurality of a third monomer that is different from the first and second monomers. This co-polymer may be used in a flow cell, and may enhance the clustering efficiency and kinetics.

First claim

What is claimed is:

1 . A co-polymer, comprising:
a plurality of a first monomer including a terminal functional group that is to attach to at least two different primers; a plurality of a second monomer including a second functional group that is different from the terminal functional group, and that is selected from the group consisting of a phenyl group, methoxy propyl, glycosyl, vinyl pyrrolidone, and an imidazole group; and a plurality of a third monomer that is different from the first and second monomers.

2 . The co-polymer as defined in claim 1 , wherein the terminal functional group is selected from the group consisting of an azide group, an amino group, an alkyne group, an aldehyde group, a hydrazine group, a carboxyl group, a hydroxyl group, a tetrazole group, a tetrazine group, a nitrile oxide group, a nitrone group, a thiol group, and combinations thereof.

3 . The co-polymer as defined in claim 2 , wherein:
the second monomer is an an acrylamide monomer selected from the group consisting of N-phenylacrylamide and N-(3-methoxypropyl)acrylamide; or the second monomer is an acrylate monomer selected from the group consisting of 2-hydroxy-1-methoxypropyl methacrylate, phenylacrylate, benzyl methacrylate, and glycosyloxyethyl methacrylate; the second monomer is the monomer containing the imidazole group, and the monomer containing the imidazole group is selected from the group consisting of 1-vinyl imidazole, 2-vinyl imidazole, and 4-vinyl imidazole,

4 . The co-polymer as defined in claim 1 , wherein:
the terminal functional group is an azide group; and the third monomer is an acrylamide.

5 . The co-polymer as defined in claim 1 , wherein:
the first monomer makes up from about 0.1% to about 20% of the co-polymer, the second monomer makes up from about 0.1% to about 20% of the co-polymer; and the third monomer makes up from about 60% to less than 100% of the co-polymer.

6 . A flow cell, comprising:
a substrate; a plurality of at least two different primers; and a co-polymer including:
a plurality of a first monomer including a terminal functional group, wherein at least some of the plurality of at least two different primers are respectively attached to at least some of the terminal functional groups;
a plurality of a second monomer including a second functional group that is different from the terminal functional group, and that is selected from the group consisting of a phenyl group, methoxy propyl, glycosyl, vinyl pyrrolidone, and an imidazole group; and
a plurality of a third monomer that is different from the first and second monomers.

7 . The flow cell as defined in claim 6 , wherein the terminal functional groups are selected from the group consisting of an azide group, an amino group, an alkyne group, an aldehyde group, a hydrazine group, a carboxyl group, a hydroxyl group, a tetrazole group, a tetrazine group, a nitrile oxide group, a nitrone group, a thiol group, and combinations thereof.

8 . The flow cell as defined in claim 7 , wherein:
the second monomer is an an acrylamide monomer selected from the group consisting of N-phenylacrylamide and N-(3-methoxypropyl)acrylamide; or the second monomer is an acrylate monomer selected from the group consisting of 2-hydroxy-1-methoxypropyl methacrylate, phenylacrylate, benzyl methacrylate, and glycosyloxyethyl methacrylate; the second monomer is the monomer containing the imidazole group, and the monomer containing the imidazole group is selected from the group consisting of 1-vinyl imidazole, 2-vinyl imidazole, and 4-vinyl imidazole.

9 . The flow cell as defined in claim 6 , wherein:
the substrate includes depressions separated by interstitial regions; and the co-polymer and the plurality of at least two different primers are positioned within at least some of the depressions.

10 . A method for improving sequencing metrics, comprising:
grafting a primer set to a polymeric hydrogel on a flow cell surface in the presence of a carbonate buffer for a time ranging from greater than 30 minutes to about 120 minutes; and amplifying a library template strand using the grafted primer set.

11 . The method as defined in claim 10 , wherein the carbonate buffer includes sodium carbonate and sodium bicarbonate.

12 . The method as defined in claim 11 , wherein a concentration of the sodium carbonate ranges from about 0.01 g/L to about 2.8 g/L and a concentration of the sodium bicarbonate ranges from about 0.09 g/L to about 2.1 g/L.

13 . A method for improving sequencing metrics, comprising:
pre-treating a flow cell including a primer set grafted to a polymeric hydrogel by: introducing water, a basic buffer having a pH ranging from 8 to 11, a high salt buffer, formamide, or isopropyl alcohol to the flow cell; increasing the flow cell to a temperature ranging from about 25° C. to about 80° C.; holding the temperature for a time ranging from about 5 minutes to about 2 hours; and then amplifying a library template strand using the grafted primer set.

14 . The method as defined in claim 13 , wherein:
the temperature of the flow cell is increased to about 60° C.; and the temperature is held for about 1 hour.

15 . The method as defined in claim 13 , wherein:
the temperature of the flow cell is increased to about 40° C.; and the temperature is held for at least 30 minutes.

16 . The method as defined in claim 13 , wherein:
the basic buffer or the high salt buffer is introduced; and the basic buffer is selected from the group consisting of a borate buffer, N-cyclohexyl-3-aminopropanesulfonic acid, 2-Amino-2-methyl-1-propanol buffer, or N-Cyclohexyl-2-aminoethanesulfonic acid; or the high salt buffer is a water base solution including above 0.1 M of a non-acidic and non-basic salt and its acid or base pair.

17 . A polymeric hydrogel, comprising:
an acrylamide co-polymer including terminal azide groups in at least some of the side chains; and a strained alkyne activated polyethylene glycol (PEG) attached to some of the terminal azide groups.

18 . The polymeric hydrogel as defined in claim 17 , wherein a concentration of the strained alkyne activated polyethylene glycol in the polymeric hydrogel ranges from about 0.5 mM to about 0.2 mM.

19 . The polymeric hydrogel as defined in claim 17 , wherein a weight average molecular weight of a PEG portion of the strained alkyne activated polyethylene glycol ranges from about 1,000 g/mol to about 20,000 g/mol.

20 . The polymeric hydrogel as defined in claim 17 , wherein the strained alkyne activated polyethylene glycol is dibenzocyclooctyne (DBCO) activated polyethylene glycol, monofluorinated cyclooctyne activated polyethylene glycol, difluorinated cyclooctyne activated polyethylene glycol, biarylazacyclooctynone activated polyethylene glycol, and bicyclo[6.1.0]nonyne activated polyethylene glycol.

Assignees

• Illumina Inc
• Illumina Cambridge Ltd

Inventors

• Artioli Gianluca Andrea
• Beech Timothy J N
• Lessard-Viger Mathieu
• Turk-Macleod Rebecca
• Mather Brian D
• Xi Weixian
• Von Hatten Xavier

Classifications

• C08F220/56 Primary

  Acrylamide; Methacrylamide · CPC title

• C08J3/075

  Macromolecular gels · CPC title

• C12Q1/6869

  Methods for sequencing · CPC title

• C08J2333/26

  Homopolymers or copolymers of acrylamide or methacrylamide · CPC title

• C12Q1/6874 Primary

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