Flow cell surface patterning

1 . A method, comprising:

applying a polymeric hydrogel to a surface of a substrate including:

a first region that is covered by an ultraviolet light blocking layer;

a second region that is adjacent to the first region and that is free of the ultraviolet light blocking layer; and

depressions separated by interstitial regions, each depression defined within at least a portion of the first region and within at least a portion of the second region;

before or after applying the polymeric hydrogel, grafting a primer set to the polymeric hydrogel to form a grafted layer, the primer set including 3′ blocked uncleavable first pre-primers and 3′ blocked uncleavable second pre-primers;

removing the polymeric hydrogel or the grafted layer from the interstitial regions;

at a first predetermined region of the grafted layer within the depression and within the second region:

altering 3′ blocked uncleavable first pre-primers to introduce cleavable first primers; and

altering 3′ blocked uncleavable second pre-primers to introduce uncleavable second primers; and

at a second predetermined region of the grafted layer within the depression and within the first region:

altering 3′ blocked uncleavable first pre-primers to introduce uncleavable first primers; and

altering 3′ blocked uncleavable second pre-primers to introduce cleavable second primers.

2 . The method as defined in claim 1 , wherein:

at the first predetermined region of the grafted layer, altering 3′ blocked uncleavable first pre-primers to introduce cleavable first primers involves:

directing ultraviolet light through the substrate, thereby removing 3′ photocleavable blocking groups i) from 3′ blocked uncleavable first pre-primers to generate unblocked uncleavable first pre-primers that overlie the second region and ii) from 3′ blocked uncleavable second pre-primers to generate unblocked uncleavable second pre-primers that overlie the second region, whereby 3′ blocked uncleavable first pre-primers and 3′ blocked uncleavable second pre-primers that overlie the first region remain blocked;

respectively hybridizing first primer generation templates to the unblocked uncleavable first pre-primers that overlie the second region and to 3′ blocked uncleavable first pre-primers that overlie the first region;

using a nucleotide mixture including a cleavable base, initiating polymerase extension along the first primer generation templates at 3′ ends of the unblocked uncleavable first pre-primers to generate the cleavable first primers; and

dehybridizing the first primer generation templates; and

at the first predetermined region of the grafted, altering the unblocked uncleavable second pre-primers to introduce uncleavable second primers involves:

respectively hybridizing second primer generation templates to the unblocked uncleavable second pre-primers that overlie the second region and to 3′ blocked uncleavable second pre-primers that overlie the first region;

using a nucleotide mixture including a thymine base, initiating polymerase extension along the second primer generation templates at 3′ ends of the unblocked uncleavable second pre-primers to generate the uncleavable second primers; and

dehybridizing the second primer generation templates.

3 . The method as defined in claim 2 , wherein:

the substrate is part of a flow cell including a second substrate opposed to the substrate, the second substrate including second depressions separated by second interstitial regions and a second ultraviolet light blocking layer positioned to block ultraviolet light directed through the second substrate from reaching the second region of the substrate and to allow ultraviolet light directed through the second substrate to reach the first region of the substrate, wherein each second depression further includes a second grafted layer applied to the second depressions, the second grafted layer including a second polymeric hydrogel and a second primer set attached thereto, the second primer set including 3′ blocked uncleavable first pre-primers and 3′ blocked uncleavable second pre-primers;

directing ultraviolet light through the substrate involves directing ultraviolet light through each of the substrate and the second substrate;

prior to directing the ultraviolet light through the substrates, the method further comprises introducing an ultraviolet light absorbing material into the flow cell; and

after directing the ultraviolet light through the substrates, the method further comprises removing the ultraviolet light absorbing material from the flow cell.

4 . The method as defined in claim 2 , further comprising directing ultraviolet light at the surface of the substrate, thereby removing 3′ photocleavable blocking groups i) from 3′ blocked uncleavable first pre-primers to generate unblocked uncleavable first pre-primers that overlie the first region and ii) from 3′ blocked uncleavable second pre-primers to generate unblocked uncleavable second pre-primers that overlie the first region.

5 . The method as defined in claim 4 , wherein:

at the second predetermined region of the grafted layer, altering 3′ blocked uncleavable first pre-primers to introduce uncleavable first primers involves:

respectively hybridizing first primer generation templates to the unblocked uncleavable first pre-primers that overlie the first region and to the cleavable first primers that overlie the second region;

initiating polymerase extension along the first primer generation templates at 3′ ends of the unblocked uncleavable first pre-primers to generate the uncleavable first primers; and

dehybridizing the first primer generation templates; and

at the second predetermined region of the grafted layer within the first region of at least some of the depressions, altering 3′ blocked uncleavable second pre-primers to introduce cleavable second primers involves:

respectively hybridizing second primer generation templates to the unblocked uncleavable second pre-primers that overlie the first region and to the uncleavable second primers that overlie the second region;

using a nucleotide mixture including a cleavable base, initiating polymerase extension along the second primer generation templates at 3′ ends of the unblocked uncleavable second pre-primers to generate the cleavable second primers; and

dehybridizing the second primer generation templates.

6 . The method as defined in claim 4 , wherein:

the substrate is part of a flow cell including a second substrate opposed to the substrate, the second substrate including second depressions separated by second interstitial regions and a second ultraviolet light blocking layer positioned to block ultraviolet light directed through the second substrate from reaching the second region of the substrate and to allow ultraviolet light directed through the second substrate to reach the first region of the substrate, wherein each second depression further includes a second grafted layer applied to the second depressions, the second grafted layer including a second polymeric hydrogel and a second primer set attached thereto, the second primer set including 3′ blocked uncleavable first pre-primers and 3′ blocked uncleavable second pre-primers; and

prior to directing the ultraviolet light at the surface of the substrate, the method further comprises removing the ultraviolet light blocking layer and the second ultraviolet light blocking layer.

7 . A kit, comprising:

a flow cell including:

a substrate including depressions separated by interstitial regions;

a polymeric hydrogel applied within each of the depressions; and

a primer set grafted to the polymeric hydrogel, the primer set including 3′ blocked uncleavable first pre-primers and 3′ blocked uncleavable second pre-primers;

first primer generation templates;

second primer generation templates;

a first nucleotide mixture including a cleavable base and uncleavable bases; and

a second nucleotide mixture including uncleavable bases.

8 . The kit as defined in claim 7 , wherein the flow cell includes an ultraviolet light blocking layer in a region of the substrate that corresponds with a predetermined region wherein 3′ blocked uncleavable first pre-primers and 3′ blocked uncleavable second pre-primers are to be altered.

9 . The method as defined in claim 6 , wherein removing the ultraviolet light blocking layer and the second ultraviolet light blocking layer involves:

wet etching the ultraviolet light blocking layer and the second ultraviolet light blocking layer, thereby creating respective gaps between the substrate and the grafted layer and between the second substrate and the second grafted layer; and

increasing adhesion between the substrate and the grafted layer and between the second substrate and the second grafted layer.

10 . The method as defined in claim 9 , where wet etching involves exposure to a 1%-2% KOH solution or a sodium carbonate buffer for about 3 to 5 minutes, without mechanical stress.