Flow cells and methods

Patent metadata

Field	Value
Publication number	US-2025207123-A1
Application number	US-202318851624-A
Country	US
Kind code	A1
Filing date	Dec 15, 2023
Priority date	Dec 16, 2022
Publication date	Jun 26, 2025
Grant date	—

Abstract

An example of a flow cell includes a substrate having depressions separated by interstitial regions: a polymeric hydrogel positioned within each of the depressions; and a plurality of transposome complexes immobilized within each of the depressions by a biotin-containing linker. In this example, each of the plurality of the transposome complexes is of a single type including a transposon end with a portion of a transferred strand hybridized to a portion of a non-transferred strand, wherein the transferred strand includes a first amplification domain and is free of an index sequence.

First claim

What is claimed is:

1 . A flow cell, comprising:
a substrate having depressions separated by interstitial regions; a polymeric hydrogel positioned within each of the depressions; and a plurality of transposome complexes immobilized within each of the depressions by a biotin-containing linker, each of the plurality of the transposome complexes being of a single type including a transposon end with a portion of a transferred strand hybridized to a portion of a non-transferred strand, wherein the transferred strand includes a first amplification domain and is free of an index sequence.

2 . The flow cell as defined in claim 1 , wherein the non-transferred strand consists of the portion hybridized to the portion of the transferred strand.

3 . The flow cell as defined in claim 1 or claim 2 , further comprising a first primer and a second primer immobilized within each of the depressions by respective second biotin-containing linkers, and wherein:
the first primer corresponds with the first amplification domain; and the second primer has a sequence that is complementary to a second amplification domain of an adapter that is to be hybridized to a portion of the transferred strand.

4 . The flow cell as defined in claim 1 , wherein:
the transferred strand and the non-transferred strand form a forked adapter; and the non-transferred strand further includes a sequencing primer sequence, an index sequence, and a second amplification domain that is complementary to a primer that is to be immobilized within each of the depressions of the flow cell.

5 . The flow cell as defined in claim 1 , further comprising a first primer and a second primer immobilized within each of the depressions by respective second biotin-containing linkers, wherein:
the first primer corresponds with the first amplification domain; the transferred strand and the non-transferred strand form a forked adapter; and the non-transferred strand further includes a sequencing primer sequence, an index sequence, and a second amplification domain that is complementary to the second primer.

6 . A tagmentation kit, comprising:
a flow cell including:
a substrate having depressions separated by interstitial regions; and
a plurality of transposome complexes immobilized within each of the depressions by a biotin-containing linker, each of the plurality of the transposome complexes being of a single type including a transposon end with a portion of a transferred strand hybridized to a non-transferred strand, wherein the transferred strand includes a first amplification domain; and
an adapter fluid including:
a carrier fluid; and
an adapter including:
a sequence complementary to the portion of the transferred strand of each of the plurality of transposome complexes;
a sequencing primer sequence;
an index sequence; and
a second amplification domain that is complementary to a primer that is to be immobilized within each of the depressions of the flow cell.

7 . The tagmentation kit as defined in claim 6 , further comprising a grafting fluid including:
a second carrier fluid; a first primer; and a second primer that is complementary to the second amplification domain, each of the first and second primers having a second biotin-containing linker at its 5′ end.

8 . The tagmentation kit as defined in claim 6 , wherein the flow cell further includes:
a first primer; and a second primer that is complementary to the second amplification domain, each of the first and second primers immobilized within each of the depressions by a second biotin-containing linker at its 5′ end.

9 . A tagmentation kit, comprising:
a flow cell including:
a substrate having depressions separated by interstitial regions; and
a plurality of transposome complexes immobilized within each of the depressions by a biotin-containing linker, each of the plurality of the transposome complexes being of a single type including a transposon end with a portion of a transferred strand hybridized to a portion of a non-transferred strand to form a forked adapter, wherein the transferred strand includes a first amplification domain, a sequencing primer sequence, an index sequence, and a second amplification domain that is complementary to a primer that is to be immobilized within each of the depressions of the flow cell; and
a grafting fluid including:
a carrier fluid;
a first primer; and
a second primer that is complementary to the second amplification domain, each of the first and second primers having a second biotin-containing linker at its 5′ end.

10 . A method, comprising:
generating at least partially adapted DNA sample fragments on a flow cell using a plurality of transposome complexes attached to a surface of the flow cell within a flow channel by a biotin-containing linker; cleaving the at least partially adapted DNA sample fragments such that the biotin-containing linkers remain attached to the surface; and one of: using the flow cell for a subsequent cycle of generating at least partially adapted DNA sample fragments; or using the flow cell to amplify at least some of the previously cleaved and at least partially adapted DNA sample fragments.

11 . The method as defined in claim 10 , wherein:
each of the plurality of the transposome complexes is of a single type including a transposon end with a portion of a transferred strand hybridized to a portion of a non-transferred strand to form a forked adapter, wherein the transferred strand includes a first amplification domain, and wherein the non-transferred strand further includes a sequencing primer sequence, an index sequence, and a second amplification domain that is complementary to a primer that is or is to be immobilized within each of the depressions of the flow cell; each of the at least partially adapted DNA sample fragments is a fully adapted DNA sample fragment; and generating the fully adapted DNA sample fragments includes:
introducing a DNA sample and a tagmentation buffer to the flow cell;
performing tagmentation of the DNA sample using the plurality of transposome complexes;
removing a transposase enzyme of each of the plurality of transposome complexes; and
performing gap fill ligation to attach the DNA sample fragments to respective adapters.

12 . The method as defined in claim 11 , wherein after cleaving the fully adapted DNA sample fragments such that the biotin-containing linker remains attached to the surface and prior to using the flow cell, the method further comprises removing streptavidin from the biotin-containing linker.

13 . The method as defined in any one of claim 10 through claim 12 , wherein the flow cell is used for the subsequent cycle of generating at least partially adapted DNA sample fragments, and wherein the subsequent cycle of generating at least partially adapted DNA sample fragments involves:
introducing a new plurality of the transposome complexes into the flow channel, whereby at least some of the new plurality of the transposome complexes attach to at least some of the biotin-containing linkers within the flow channel; introducing a new DNA sample and a new tagmentation buffer to the flow channel; and repeating the tagmentation, transposase enzyme removal, and gap fill ligation with the new DNA sample.

14 . The method as defined in claim 11 or claim 12 , wherein the flow cell is used for the amplification of the previously cleaved and fully adapted DNA sample fragments, and wherein the amplification of the previously cleaved and fully adapted DNA sample fragments involves:
attaching first and second primers to at least some of the biotin-cont

Assignees

• Illumina Inc

Inventors

• Artioli Gianluca Andrea
• Basuki Johan Sebastian
• Ciesla Craig
• Dill Tyler J
• Fraser Louise Jane
• Gormley Niall Anthony
• Ho Yuan Heng Trevor
• Karunakaran Aathavan
• Martins Vitoriano Maria Ines
• Moore Jessica
• Ricoult Sebastien Georg Gabriel
• Thomson Vicki S
• Von Hatten Xavier
• Weir Jacqueline C

Classifications

• G01N2333/91245

  Nucleotidyltransferases (2.7.7) · CPC title

• C12Q1/6855

  Ligating adaptors · CPC title

• C12Q1/6806 Primary

  Preparing nucleic acids for analysis, e.g. for polymerase chain reaction [PCR] assay (C12Q1/6804 takes precedence) · CPC title

• C12Q1/485

  involving kinase · CPC title

• C12N15/1093

  General methods of preparing gene libraries, not provided for in other subgroups · CPC title