Granted Patent
B2
US 12,570,971 · App. 17/719,276 · Granted Mar 10, 2026
Contiguity preserving transposition
Inventors:
Frank J. Steemers (San Diego, CA); Kevin L. Gunderson (San Diego, CA); Fan Zhang (San Diego, CA); Jason Richard Betley (Essex, GB); Niall Anthony Gormley (Essex, GB); Wouter Meuleman (San Diego, CA); Jacqueline Weir (Essex, GB); Avgousta Ioannou (Essex, GB); Gareth Jenkins (Essex, GB); Rosamond Jackson (Essex, GB); Natalie Morrell (Essex, GB); Dmitry K. Pokholok (San Diego, CA); Steven J. Norberg (San Diego, CA); Molly He (San Diego, CA); Amirali Kia (San Diego, CA); Igor Goryshin (Madison, WI); Rigo Pantoja (San Diego, CA)
Assignee:
Illumina Cambridge Limited
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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.
Claims (17)
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.
Assignments (1)
ASSIGNMENT OF ASSIGNOR'S INTEREST
Recorded May 13, 2022
From: 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; HE, MOLLY; KIA, AMIRALI; GORYSHIN, IGOR; PANTOJA, RIGO
To: ILLUMINA CAMBRIDGE LIMITED
Reel/Frame 059906/0206 →
Continuity (6)
Continuation
15519482
Provisional Application
62242880
· Oct 16, 2015
Provisional Application
62157396
· May 5, 2015
Provisional Application
62065544
· Oct 17, 2014
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