US 2026/0308571 A1 · Contiguity Preserving Transposition

Contiguity Preserving Transposition

AssigneeIllumina Cambridge Limited

InventorsFrank J. Steemers, Kevin L. Gunderson, Fan Zhang, Jason Richard Betley, Niall Anthony Gormley, Wouter Meuleman, Jacqueline Weir, Avgousta Ioannou, Gareth Jenkins, Rosamond Jackson, Natalie Morrell, Dmitry K. Pokholok, Steven J. Norberg, Molly He, Amirali Kia, Igor Goryshin, Rigo Pantoja

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Abstract

Embodiments provided herein relate to methods and compositions for preparing an immobilized library of barcoded DNA fragments of a target nucleic acid, identifying genomic variants, determining the contiguity information, phasing information, and methylation status of the target nucleic acid.

Claims21

Dependent claims

1-20. (canceled)

A method of preparing a sequencing library for determining a methylation status of a target nucleic acid comprising:

fragmenting the target nucleic acid into two or more fragments to generate target nucleic acid fragments;
incorporating a first common adaptor sequence into the 5′-ends of the target nucleic acid fragments, wherein the first common adaptor sequence comprises a first primer binding sequence and an affinity moiety, wherein the affinity moiety is a first member of a binding pair;
denaturing the target nucleic acid fragments to generate single stranded target nucleic acid fragments;
immobilizing the single stranded target nucleic acid fragments on a solid support to generate immobilized target nucleic acid fragments, wherein the solid support comprises a second member of the binding pair to permit immobilization of an individual target nucleic acid fragment via binding between the first member and the second member of the binding pair;
subjecting the immobilized target nucleic acid fragments to bisulfite treatment;
incorporating a second common adaptor sequence to the bisulfite treated immobilized target nucleic acid fragments, wherein the second common adaptor sequence comprises a second primer binding sequence;
and
amplifying the bisulfite treated target nucleic acid fragments immobilized on the solid support using a first and a second primer, thereby producing a sequencing library for determining the methylation status of the target nucleic acid.

5 more elements

A method of preparing a sequencing library for determining a methylation status of a target nucleic acid comprising:

fragmenting the target nucleic acid into two or more fragments to generate target nucleic acid fragments;
incorporating a first common adaptor sequence into the 5′-ends of the target nucleic acid fragments, wherein the first common adaptor sequence comprises a first primer binding sequence and an affinity moiety, wherein the affinity moiety is a first member of a binding pair;
denaturing the target nucleic acid fragments to generate single stranded nucleic acid fragments;
immobilizing the single stranded target nucleic acid fragments on a solid support to generate immobilized target nucleic acid fragments, wherein the solid support comprises a second member of the binding pair to permit immobilization of an individual target nucleic acid fragment via binding between the first member and the second member of the binding pair;
subjecting the immobilized target nucleic acid fragments to bisulfite treatment;
incorporating a second common adaptor sequence to the bisulfite treated immobilized target nucleic acid fragments, wherein the second common adaptor sequence comprises a second primer binding sequence;
and
extending the 3′-end of the immobilized target nucleic acid fragments such that the 3′-end of the immobilized target nucleic acid fragments comprise a homopolymeric tail;
and
amplifying the bisulfite treated target nucleic acid fragments immobilized on the solid support, thereby producing a sequencing library for determining the methylation status of the target nucleic acid.

7 more elements

Dependencies are inferred from the claim text.