Granted Patent B2

US 12,497,713 · App. 17/051,870 · Granted Dec 16, 2025

Inventors: Radoje T. Drmanac (San Jose, CA); Brock A. Peters (San Jose, CA); Ou Wang (Shenzhen, CN)

Assignees: MGI Tech Co., Ltd.; BGI Shenzhen

C40B50/16C12N15/1082C40B20/04

Abstract

Claims (39)

1 . A method of preparing a sequencing library for sequencing a target nucleic acid without the use of nanodrops, comprising:

(a) transposing an insertion sequence in a transposome into first fragments derived from the target nucleic acid while the transposome is in solution, wherein the insertion sequence comprises a hybridization sequence, and wherein the transposing produces nicks in the first fragments and 3′ hydroxyl at said nicks; (b) combining in a single vessel or mixture (i) the first fragments of the target nucleic acid from (a), (ii) a splint oligonucleotide, and (iii) a population of beads, wherein each bead comprises capture oligonucleotides immobilized thereon, said capture oligonucleotides comprising

1) A barcode-containing sequence, wherein the capture oligonucleotides immobilized on the same individual bead comprise the same barcode-containing sequence and a majority of beads have different barcode-containing sequences,

2) A common sequence complementary to at least a portion of the splint oligonucleotide, where a second portion of the splint oligonucleotide is complementary to at least a portion of the hybridization sequence;

(c) ligating capture oligonucleotides of individual beads to inserted hybridization sequences of individual first fragments; and

 (d) ligating a 3′ branch ligation adaptor oligonucleotide to the first fragments at the nicks via a 3′ branch ligation, wherein 3′ branch ligation adaptor oligonucleotide is a blunt end adaptor and 3′ branch ligation comprises joining of 5′ phosphate from the blunt-end adapter covalently to the 3′ hydroxyl at the nicks of the first fragments.

2 . The method of claim 1 wherein the capture oligonucleotides comprise a first PCR primer annealing site,

and wherein 3′ branch ligation adaptor oligonucleotide comprises a second PCR primer annealing site.

3 . The method of claim 2 wherein the first PCR primer annealing site and the second PCR primer annealing site have different sequences.

4 . The method of claim 2 wherein 3′ branch ligation adaptor oligonucleotide comprises a tag sequence.

5 . The method of claim 1 wherein in the step of transposing the insertion sequence into the first fragments a transposase enzyme remains bound to the first fragments; wherein the method further comprises

(d) removing the transposase from the first fragments thereby producing subfragments.

6 . The method of claim 5 comprising amplifying the subfragments to produce amplicons.

7 . The method of claim 6 comprising sequencing the amplicons to produce sequence reads wherein sequence reads with the same barcode-containing sequence are from the same first fragment.

8 . The method of claim 1 wherein the target nucleic acid is human genomic DNA.

9 . The method of claim 1 wherein two different insertion sequences are inserted into the first fragments.

10 . The method of claim 1 wherein the target nucleic acid is not amplified prior to step (a).

11 . The method of claim 1 wherein the beads comprise at least 100,000 copies of the capture oligonucleotide.

12 . The method of claim 6 wherein at least some capture oligonucleotides are enzymatically removed using exonuclease, optionally exonuclease I or III or both.

13 . The method of claim 1 wherein the capture oligonucleotides and/or the splint oligonucleotides comprise uracil such that treatment with a uracil-DNA glycosylase (UDG) degrades uracil-containing oligonucleotides.

14 . The method of claim 1 wherein >90% of the first fragments are barcoded.

15 . The method of claim 5 wherein >90% of subfragments in a fragment are ligated to capture oligonucleotide.

16 . The method of claim 1 wherein, on average, the spacing of the capture oligonucleotides on the beads is <30 nm.

17 . The method of claim 7 wherein >50% of the subfragments are sequenced.

18 . The method of claim 7 further comprising:

(e) assigning a majority of the sequence reads to corresponding first fragments; and

(f) assembling the sequence reads to produce an assembled sequence of the target nucleic acid.

19 . The method of claim 1 wherein the majority of first fragments in step (a) are longer than 20 kb.

20 . The method of claim 1 wherein a majority of the first fragments are in a range of from 50 kilobases to 200 kilobases in length.

21 . The method of claim 1 wherein the first fragments are from a single cell.

22 . The method of claim 1 wherein the first fragments in the single mixture contains 5-100 genome equivalents of human DNA.

23 . The method of claim 1 in which the population of beads comprises, in aggregate, at least 100,000 different tag sequences.

24 . The method of claim 1 , wherein the first fragments are duplex DNA or DNA/RNA hybrid molecules.

25 . The method of claim 1 , wherein ligating the first oligonucleotide to the first fragments is by a T4 DNA ligase.

26 . The method of claim 1 , wherein first fragments are concatemers of DNA.

27 . The method of claim 1 , wherein a plurality of first fragments are from a single cell.

28 . The method of claim 1 , wherein, on average, the spacing of the adaptor oligonucleotide on the beads is <10 nm, <15 nm, <20 nm, <30 nm, <40 nm, or 50 nm.

29 . The method of claim 1 , wherein the population of beads comprises 10-50 million beads.

30 . The method of claim 1 , wherein the beads have a diameter in a range of 2 μm-8 μm.

Assignments (5)

Continuity (4)

References Cited (211)