US 11,873,480 B2
Contiguity preserving transposition
Frank J. Steemers, San Diego, CA (US); Kevin L. Gunderson, San Diego, CA (US); Fan Zhang, San Diego, CA (US); Jason Richard Betley, Essex (GB); Niall Anthony Gormley, Essex (GB); Wouter Meuleman, San Diego, CA (US); 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 (US); Steven J. Norberg, San Diego, CA (US); Molly He, San Diego, CA (US); Amirali Kia, San Diego, CA (US); Igor Goryshin, Madison, WI (US); and Rigo Pantoja, San Diego, CA (US)
Assigned to ILLUMINA CAMBRIDGE LIMITED, Cambridgeshire (GB)
Appl. No. 15/519,482
Filed by Illumina Cambridge Limited, Essex (GB)
PCT Filed Oct. 16, 2015, PCT No. PCT/US2015/056040
§ 371(c)(1), (2) Date Apr. 14, 2017,
PCT Pub. No. WO2016/061517, PCT Pub. Date Apr. 21, 2016.
Claims priority of provisional application 62/242,880, filed on Oct. 16, 2015.
Claims priority of provisional application 62/157,396, filed on May 5, 2015.
Claims priority of provisional application 62/065,544, filed on Oct. 17, 2014.
Prior Publication US 2019/0040382 A1, Feb. 7, 2019
Int. Cl. C12N 15/10 (2006.01); C12Q 1/6806 (2018.01); C12Q 1/6874 (2018.01); C12Q 1/6876 (2018.01)
CPC C12N 15/1065 (2013.01) [C12N 15/1082 (2013.01); C12N 15/1093 (2013.01); C12Q 1/6806 (2013.01); C12Q 1/6874 (2013.01); C12Q 1/6876 (2013.01); C12Q 2600/154 (2013.01); C12Q 2600/156 (2013.01); C12Q 2600/172 (2013.01)] 23 Claims
 
1. A method of preparing a library of barcoded DNA fragments of a target DNA comprising:
a. binding a target DNA to a plurality of transposome complexes, each transposome complex comprising transposons and transposases,
wherein the transposons comprise transferred strands and non-transferred strands, and
wherein at least one of the transposons of the transposome complex comprises an adaptor sequence capable of hybridizing to a complementary capture sequence;
b. fragmenting the target DNA of step a into a plurality of contiguously-linked, transposed DNA fragments and inserting a plurality of transferred strands into at least one strand of each contiguously-linked transposed DNA fragment of the plurality of contiguously-linked transposed fragments, wherein contiguity of DNA fragments of the target DNA is maintained by the transposases;
c. immobilizing the plurality of contiguously-linked, transposed DNA fragments of step b on a plurality of solid supports by hybridizing the adaptor sequence of the at least one of the transposons to a complementary capture sequence, each of the solid supports in the plurality comprising a plurality of immobilized oligonucleotides, each oligonucleotide of the plurality of immobilized oligonucleotides comprising, in sequential order extending from a surface of the solid support:
(i) a first primer binding site attached to the solid support;
(ii) a first barcode sequence; and
(iii) a complementary capture sequence capable of hybridizing to the adaptor sequence; and
d. attaching the first barcode sequence to one or more of the plurality of contiguously-linked, transposed DNA fragments of step c, thereby producing a library of barcoded, linked DNA fragments,
wherein the first barcode sequences of all of the oligonucleotides of the plurality of oligonucleotides immobilized on a given solid support comprise the same nucleic acid sequence,
wherein a nucleic acid sequence of the first barcode sequence of the oligonucleotides of the plurality of oligonucleotides immobilized on a given solid support in the plurality of the solid supports differs from a nucleic acid sequence of all of the first barcode sequences from other solid supports in the plurality of solid supports, and
wherein the steps (a)-(d) are carried out in a single reaction compartment.