Massively parallel contiguity mapping

Patent metadata

Field	Value
Publication number	US-11299730-B2
Application number	US-201916665800-A
Country	US
Kind code	B2
Filing date	Oct 28, 2019
Priority date	Feb 2, 2011
Publication date	Apr 12, 2022
Grant date	Apr 12, 2022

Abstract

Contiguity information is important to achieving high-quality de novo assembly of mammalian genomes and the haplotype-resolved resequencing of human genomes. The methods described herein pursue cost-effective, massively parallel capture of contiguity information at different scales.

First claim

What is claimed is:

1. A method of preparing a sequencing library, comprising:
(a) contacting a target DNA molecule with a plurality of transposases to insert continuous transposons at a plurality of internal locations in the target DNA molecule to produce a modified target DNA molecule, wherein the continuous transposons each comprise a first flowcell sequence corresponding to a first surface-bound flowcell primer;
(b) contacting the modified target DNA molecule produced in step (a) with a flowcell to allow hybridization of the first flowcell sequences in the plurality of internally inserted continuous transposons to a plurality of copies of the first surface-bound flowcell primer at co-linear coordinates on the flowcell;
(c) performing cluster amplification of a plurality of subsequences of the target DNA molecule on the flowcell to produce a plurality of clusters at the co-linear coordinates on the flowcell, wherein each of the plurality of subsequences is adjacent to one of the plurality of internally inserted continuous transposons within the modified target DNA molecule produced in step (a); and
(d) capturing contiguity information by associating sequences of the plurality of subsequences derived from the plurality of clusters at the co-linear coordinates on the flowcell with positions along an axis of the target DNA molecule.

2. The method of claim 1 , further comprising sequencing the plurality of subsequences of the target DNA molecule thereby producing the sequences.

3. The method of claim 1 , wherein the plurality of transposons are inserted at a density of about one transposon per every 35 bases to about transposon per every 600 bases.

4. The method of claim 1 , further comprising, before step (b), attaching a flowcell-compatible end adaptor to each end of the target DNA molecule wherein each of the end adaptors comprises a flowcell sequence that hybridizes to a surface-bound flowcell primer.

5. The method of claim 4 , wherein the flowcell sequence is the first flowcell sequence and the surface-bound flowcell primer is the first surface-bound flowcell primer.

6. The method of claim 4 , wherein step (b) comprises stretching the modified target DNA molecule along the flowcell surface.

7. The method of claim 6 , wherein the modified target DNA molecule comprising the added end adaptors is stretched under flow or an electric field.

8. The method of claim 1 , wherein the contiguity information comprises a positional order of the plurality of sequences obtained from the target DNA molecule.

9. The method of claim 1 , wherein the contiguity information comprises a physical distance between a pair of sequences within the target DNA molecule.

10. The method of claim 9 , wherein the distance between a pair of co-linear cluster positions on the flowcell surface is directly proportional to the distance between corresponding sequence positions along the axis of the target DNA molecule.

11. The method of claim 1 , further comprising performing the method for a plurality of target DNA molecules on the same flowcell.

12. The method of claim 1 , wherein insertion of the continuous transposons at internal locations in the target DNA molecule results in a plurality of single-stranded bubble structures at the internal locations in the target DNA molecule.

13. The method of claim 1 , wherein the plurality of internal locations in the target DNA molecule are randomly determined.

14. The method of claim 1 , wherein prior to step (a) high molecular weight DNA is sheared followed by end repair to produce the target DNA molecule with blunt ends.

15. The method of claim 14 , wherein the blunt ends are A-tailed.

16. The method of claim 1 , wherein prior to step (a) high molecular weight DNA is sheared followed by end repair to produce the target DNA molecule with blunt ends or digested with a restriction enzyme, and the method further comprises ligating a flowcell-compatible end adaptor to each end of the target DNA molecule.

17. The method of claim 16 , wherein each of the end adaptors comprises a flowcell sequence that hybridizes to a surface-bound flowcell primer.

18. The method of claim 17 , the flowcell sequence is the first flowcell sequence and the surface-bound flowcell primer is the first surface-bound flowcell primer.

Assignees

• Univ Washington Through Its Center For Commercialization

Inventors

• Shendure Jay Ashok
• Schwartz Jerrod Joseph
• Adey Andrew Colin
• Lee Cho Li
• Hiatt Joseph Brian
• Kitzman Jacob Otto
• Kumar Akash

Classifications

• C12N15/1093 Primary

  General methods of preparing gene libraries, not provided for in other subgroups · CPC title

• Y02P20/582

  Recycling of unreacted starting or intermediate materials · CPC title