Sample preparation on a solid support

• US 9,683,230 B2
• Filed: 03/08/2013
• Issued: 06/20/2017
• Est. Priority Date: 01/09/2013
• Status: Active Grant

• Current Assignee: Illumina Cambridge Limited (Illumina Incorporated)
• Original Assignee: Illumina Cambridge Limited (Illumina Incorporated)
• Inventors: Gormley, Niall Anthony, Smith, Geoffrey Paul
• Primary Examiner(s): Zhang, Kaijiang
• Application Number:
  US13/790,220
• Publication Number:
  US 20140194324A1
• Time in Patent Office: 1,565 Days
• Field of Search: None
• CPC Class Codes: B01J 19/0046 Sequential or parallel reactions, e.g. for the synthesis of polypeptides or polynucleotides; Apparatus and devices for combinatorial chemistry or for making molecular arrays synthesis methods per se C40B50/00
• CPC Class Codes: B01J 2219/00286 Reactor vessels with top and bottom openings
• CPC Class Codes: B01J 2219/00608 DNA chips
• CPC Class Codes: B01J 2219/00722 Nucleotides
• CPC Class Codes: C12N 15/1065 Preparation or screening of tagged libraries, e.g. tagged microorganisms by STM-mutagenesis, tagged polynucleotides, gene tags
• CPC Class Codes: C12N 15/1093 General methods of preparing gene libraries, not provided for in other subgroups
• CPC Class Codes: C12Q 1/6834 Enzymatic or biochemical coupling of nucleic acids to a solid phase
• CPC Class Codes: C12Q 1/6869 Methods for sequencing
• CPC Class Codes: C12Q 1/6874 involving nucleic acid arrays, e.g. sequencing by hybridisation
• CPC Class Codes: C12Q 2521/543 Immobilised enzyme(s)
• CPC Class Codes: C12Q 2525/191 incorporating an adaptor
• CPC Class Codes: C12Q 2535/122 Massive parallel sequencing
• CPC Class Codes: C12Q 2565/518 characterised by the immobilisation of the nucleic acid sample or target
• CPC Class Codes: C40B 40/08 Libraries containing RNA or DNA which encodes proteins, e.g. gene libraries
• CPC Class Codes: C40B 50/14 Solid phase synthesis, i.e. wherein one or more library building blocks are bound to a solid support during library creation; Particular methods of cleavage from the solid support
• CPC Class Codes: C40B 50/18 using a particular method of attachment to the solid support

Abstract

Presented are methods and compositions for using immobilized transposase and a transposon end for generating an immobilized library of 5′-tagged double-stranded target DNA on a surface. The methods are useful for generating 5′- and 3′-tagged DNA fragments for use in a variety of processes, including massively parallel DNA sequencing.

Claims

1. A method of preparing an immobilized library of tagged DNA fragments comprising:

(a) providing a solid support having transposome complexes immobilized thereon, wherein said transposome complexes comprise a transposase bound to a first polynucleotide, said first polynucleotide comprising

(i) a 3′

portion comprising a transposon end sequence, and

(ii) a first tag comprising a first tag domain;

(b) providing a sample comprising target DNA, proteins and other cellular components from an in vivo source;

(c) applying said sample to the solid support under conditions wherein said target DNA, proteins and other cellular components are present at the same proportion as in said in vivo source, whereby the target DNA is fragmented by the transposome complexes, and the 3′

transposon end sequence of the first polynucleotide is transferred to a 5′

end of at least one strand of the fragments;

thereby producing an immobilized library of double-stranded fragments wherein at least one strand is 5′

-tagged with the first tag.

2. The method of claim 1, wherein said first polynucleotide is immobilized to said solid support.

3. The method of claim 1, wherein said transposome complexes comprise a second polynucleotide comprising a region complementary to said transposon end sequence.

4. The method of claim 1, further comprising washing the solid support to remove any unbound nucleic acids.

5. The method of claim 1, wherein the transposome complexes are present on the solid support at a density of at least 10³, 10⁴, 10⁵, or 10⁶ complexes per mm².

6. The method of claim 1, wherein said transposome complex comprises a hyperactive Tn5 transposase.

7. The method of claim 1, wherein the lengths of the double-stranded fragments in said immobilized library are adjusted by increasing or decreasing the density of transposome complexes present on said solid support.

8. The method of claim 1, wherein at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% of the tags present on said solid support comprise the same tag domain.

9. The method of claim 1, further comprising:

(d) providing transposome complexes in solution and contacting said transposome complexes in solution with the immobilized fragments under conditions whereby the target DNA is further fragmented by the transposome complexes in solution;

thereby obtaining immobilized nucleic acid fragments having one end in solution.

10. The method of claim 9, wherein the transposome complexes in solution comprise a second tag, thereby generating immobilized nucleic acid fragments having a second tag in solution.

11. The method of claim 10, wherein the first and second tags are different.

12. The method of claim 9, wherein at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% of said transposome complexes in solution comprise a second tag comprising a second tag domain.

13. The method of claim 9, further comprising amplifying the fragments on said solid support by providing a polymerase and an amplification primer corresponding to a portion of the first polynucleotide.

14. The method of claim 1, wherein said tag domain comprises a region for cluster amplification.

15. The method of claim 1, wherein said tag domain comprises a region for priming a sequencing reaction.

16. The method of claim 1, wherein the solid support comprises microparticles.

17. The method of claim 1, wherein the solid support comprises a patterned surface.

18. The method of claim 1, wherein the solid support comprises wells.

19. The method of claim 1, wherein said target DNA is double stranded, wherein said transposition reaction and introduction of tags into said target DNA results in double stranded bridged structures across said solid support.

20. The method of claim 19, wherein the length of the bridged fragments is between 100 base pairs and 1500 base pairs.

21. The method of claim 1, wherein said sample that is applied to said solid support is blood.

22. The method of claim 1, wherein the solid support is inner or outer surface of a tube.

23. The method of claim 1, wherein said sample that is applied to said solid support is a cell lysate.

24. The method of claim 23, wherein said cell lysate is a crude cell lysate.

25. The method of claim 1, wherein said sample that is applied to said solid support has a 260/280 absorbance ratio that is less than 1.7.

26. The method of claim 1, further comprising lysing cells in said sample after applying said sample to said solid support.

27. A solid support having a library of tagged DNA fragments immobilized thereon prepared according to the method of claim 1.