Contiguity preserving transposition

Patent metadata

Field	Value
Publication number	US-12570971-B2
Application number	US-202217719276-A
Country	US
Kind code	B2
Filing date	Apr 12, 2022
Priority date	Oct 17, 2014
Publication date	Mar 10, 2026
Grant date	Mar 10, 2026

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.

First claim

What is claimed is:

1 . A method for maintaining contiguity information of a double-stranded target nucleic acid, the method comprising:
(a) providing a plurality of solid supports each having a plurality of transposome complexes immobilized thereon, wherein the plurality of transposome complexes each comprises more than one transposome monomeric unit, wherein a first transposome monomeric units is linked to a second transposome monomeric unit to form an individual transposome complex, and wherein an individual transposome monomeric unit comprises a transposase bound to a polynucleotide, said polynucleotide comprising:
(i) a 3′ portion comprising a transposon end sequence, and
(ii) a first adaptor comprising a first barcode sequence;
(b) applying double-stranded target nucleic acids to the plurality of solid supports, wherein the plurality of transposome complexes on a respective solid support of the plurality of solid supports bind to a plurality of portions of an individual double-stranded target nucleic acid under conditions whereby the individual double-stranded target nucleic acid is fragmented into double-stranded target nucleic acid fragments by the plurality of transposome complexes such that the contiguity of the individual double-stranded target nucleic acid is maintained, wherein the transposon end sequence of the polynucleotide is transferred to a 5′ end of at least one strand of the double-stranded target nucleic acid fragments, thereby producing an immobilized library of double-stranded target nucleic acid fragments wherein at least the one strand is 5′-tagged with the first barcode sequence, wherein a plurality of first barcode sequences on the respective solid support comprise the same nucleic acid sequence, and wherein a nucleic acid sequence of the plurality of first barcode sequences on the respective solid support differs from a nucleic acid sequence of a plurality of first barcode sequences from other solid supports of the plurality of solid supports; (c) determining a sequence based on the double-stranded target nucleic acid fragments and associated first barcode sequences; and (d) determining the contiguity information of the double-stranded target nucleic acids by identifying the first barcode sequences.

2 . The method of claim 1 , wherein the contiguity information of the double-stranded target nucleic acid is indicative of genomic variants selected from the group consisting of deletions, translocations, interchromosomal gene fusions, duplications, and paralogs.

3 . The method of claim 1 , wherein the double-stranded target nucleic acid fragments comprising the first barcodes are amplified prior to determining the sequence of the target nucleic acid fragments.

4 . The method of claim 1 , wherein plurality of solid supports comprises beads.

5 . The method of claim 1 , wherein the first adaptor further comprises a first primer binding sequence.

6 . The method of claim 1 , wherein the double-stranded target nucleic acid is from a single cell.

7 . The method of claim 1 , wherein the double-stranded target nucleic acid is from a single organelle.

8 . The method of claim 1 , wherein the double-stranded target nucleic acid comprises genomic DNA.

9 . The method of claim 1 , wherein the double-stranded target nucleic acid is cross-linked to another nucleic acid.

10 . The method of claim 1 , wherein the double-stranded target nucleic acid comprises cell-free tumor DNA.

11 . The method of claim 1 , wherein the double-stranded target nucleic acid comprises cDNA.

Assignees

• Illumina Cambridge Ltd

Inventors

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

Classifications

• C12Q2600/172

  Haplotypes · CPC title

• C12Q2600/156

  Polymorphic or mutational markers · CPC title

• C12Q2600/154

  Methylation markers · CPC title

• C12Q1/6876

  Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes · CPC title

• C12Q1/6874

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