High throughput genome sequencing on DNA arrays

• US 8,722,326 B2
• Filed: 10/31/2007
• Issued: 05/13/2014
• Est. Priority Date: 02/24/2006
• Status: Active Grant

• Current Assignee: Complete Genomics Incorporated (BGI Genomics Co., Ltd.)
• Original Assignee: Callida Genomics, Inc. (SBH Genomics, Inc.)
• Inventors: Drmanac, Radoje T., Callow, Matthew, Drmanac, Snezana
• Primary Examiner(s): FORMAN, BETTY J
• Application Number:
  US11/981,661
• Publication Number:
  US 20090005252A1
• Time in Patent Office: 2,386 Days
• Field of Search: None
• US Class Current: 435/6.1
• CPC Class Codes: C12Q 1/6837 using probe arrays or probe chips C12Q1/6874 takes precedence
• CPC Class Codes: C12Q 1/6874 involving nucleic acid arrays, e.g. sequencing by hybridisation
• CPC Class Codes: C12Q 2521/313 Type II endonucleases, i.e. cutting outside recognition site
• CPC Class Codes: C12Q 2525/151 repeat or repeated sequences, e.g. VNTR, microsatellite, concatemer
• CPC Class Codes: C12Q 2531/125 Rolling circle

Abstract

The present invention is directed to methods and compositions for acquiring nucleotide sequence information of target sequences using adaptors interspersed in target polynucleotides. The sequence information can be new, e.g. sequencing unknown nucleic acids, re-sequencing, or genotyping. The invention preferably includes methods for inserting a plurality of adaptors at spaced locations within a target polynucleotide or a fragment of a polynucleotide. Such adaptors may serve as platforms for interrogating adjacent sequences using various sequencing chemistries, such as those that identify nucleotides by primer extension, probe ligation, and the like. Encompassed in the invention are methods and compositions for the insertion of known adaptor sequences into target sequences, such that there is an interruption of contiguous target sequence with the adaptors. By sequencing both “upstream” and “downstream” of the adaptors, identification of entire target sequences may be accomplished.

Claims

1. A method of identifying a nucleotide sequence of a target nucleic acid, said method comprising:

(a) providing a plurality of constructs disposed on a surface of a substrate, wherein each of said plurality of constructs comprises multiple copies of a monomer and each monomer comprises a first adaptor, a second adaptor, and fragments of the target nucleic acid and has been formed by a process that comprises;

(i) forming a first circular DNA comprising a fragment of said target nucleic acid and a first adaptor, wherein the first adaptor comprises a binding site for a restriction enzyme that cleaves DNA at a cleavage site separated from said binding site by at least six nucleotides;

(ii) forming a linearized DNA by a process that comprises cleaving the first circular DNA at a site that is internal to the fragment of the target nucleic acid using a restriction enzyme that binds to said binding site; and

(iii) forming a second circular DNA comprising said linearized DNA and a second adaptor thereby forming said monomer;

then

(b) identifying a nucleotide sequence on either side of the second adaptor in a plurality of the constructs in the array.

2. The method of claim 1, wherein step (b) comprises identifying nucleotide sequence of the target nucleic acid both upstream and downstream from the second adaptor in a plurality of the constructs in the array using sequencing probes anchored on either side of the second adaptor.

3. The method of claim 1, wherein said surface comprises silica.

4. The method of claim 3, wherein said silica comprises functional moieties.

5. The method of claim 4, wherein said functional moieties are a member selected from:

amines, silanes and hydroxyl.

6. The method of claim 5, wherein said functional moieties are amines.

7. The method of claim 6, wherein said constructs are immobilized on said surface through interaction with said amines.

8. The method of claim 1, wherein said plurality of constructs is immobilized on said surface through noncovalent interactions.

9. The method of claim 1, wherein at least one of said first and second adaptor comprises a Type IIs endonuclease recognition site.

10. The method of claim 1, wherein at least one of said first and second adaptor comprises a palindromic sequence.

11. The method of claim 1, wherein said target nucleic acid is genomic DNA.

15. The method of claims 1 and 12, wherein said restriction enzyme cleaves said fragment within 20 nucleotides from said recognition site.

16. The method of claims 1 and 12, wherein said restriction enzyme cleaves said fragment at a distance of 18 to 20 nucleotides from said recognition site.

17. The method of claim 1, wherein the constructs are disposed on discrete regions on the substrate, wherein surface of the substrate between the discrete regions does not bind the constructs.

20. The method of claim 17, wherein the area of each discrete region corresponds to the size of a single concatemer.

22. The method of claim 1 or 12, wherein step (b) comprises:

(i) hybridizing an anchor probe to a probe hybridization site in the second adaptor;

(ii) hybridizing a sequencing probe to the fragment of the target nucleic acid adjacent to the hybridized anchor probe;

then

(iii) ligating the sequencing probe to the anchor probe; and

(iv) detecting the probe ligated in step (iii) to identify said nucleotide sequence in the target nucleic acid.

23. The method of claim 22, wherein steps (i) through (iv) are repeated to identify multiple nucleotide sequences of the target nucleic acid.

24. The method of claim 1, wherein step (b) comprises hybridizing two anchor probes to the second adaptor so as to determine nucleotide sequence of the target nucleic acid both upstream and downstream from the second adaptor.

25. The method of claim 1, wherein the surface comprises more than 100,000 concatemers per square millimeter.

26. The method of claim 1, wherein at least 70% of said concatemers are optically resolvable.

27. The method of claim 1, wherein each discrete spaced apart region has an area of less than 1 μ

m².

12. A method of identifying a nucleotide sequence of a target nucleic acid, said method comprising:

(a) providing a substrate comprising a plurality of immobilized concatemers, wherein said substrate comprises a plurality of discrete regions, and wherein said concatemers are randomly disposed on said discrete regions, wherein each concatemer is formed by replication of a monomer such that said concatemer comprises multiple copies of said monomer, and wherein said monomer comprises a first adaptor, a second adaptor, and fragments of the target nucleic acid and has been formed by a process comprising;

(i) forming a first circular DNA comprising a fragment of said target nucleic acid and a first adaptor, wherein the first adaptor comprises a binding site for a restriction enzyme that cleaves DNA at a cleavage site separated from said binding site by at least six nucleotides;

(ii) forming a linearized DNA by a process that comprises cleaving the first circular DNA at a site that is internal to the fragment of the target nucleic acid using a restriction enzyme that binds to said binding site;

(iii) forming a second circular DNA comprising said linearized DNA and a second adaptor thereby forming said monomer; and

(iv) replicating the second circular DNA to form said concatemer;

then

(b) determining nucleotide sequence of the target nucleic acid both upstream and downstream from the second adaptor in a plurality of the constructs in the array.

13. The method of claim 12, wherein steps (ii) and (iii) are repeated a number of times.

14. The method of claim 12, wherein said concatemers are attached to said discrete regions through a noncovalent interaction.

18. The method of claim 12 or 17, wherein said discrete regions have a size from about 125 nm to about 250 nm.

19. The method of claim 12 or 17, wherein said discrete regions have a size from about 200 nm to about 500 nm.

21. The method of claim 12, wherein the area of each discrete region corresponds to the size of a single concatemer.