High throughput genome sequencing on DNA arrays

• US 20090264299A1
• Filed: 02/26/2007
• Published: 10/22/2009
• Est. Priority Date: 02/24/2006
• Status: Abandoned Application

• Current Assignee: Callida Genomics, Inc. (SBH Genomics, Inc.)
• Original Assignee: Complete Genomics Incorporated (BGI Genomics Co., Ltd.)
• Inventors: Drmanac, Radoje, Drmanac, Snezana, Callow, Matthew J.
• Application Number:
  US11/679,124
• Publication Number:
  US 20090264299A1
• Time in Patent Office: Days
• US Class Current: 506/3
• CPC Class Codes: C12N 15/64 General methods for preparing the vector, for introducing it into the cell or for selecting the vector-containing host
• CPC Class Codes: C12N 15/66 General methods for inserting a gene into a vector to form a recombinant vector using cleavage and ligation; Use of non-functional linkers or adaptors, e.g. linkers containing the sequence for a restriction endonuclease
• 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 2525/131 incorporating a restriction site
• CPC Class Codes: C12Q 2525/151 repeat or repeated sequences, e.g. VNTR, microsatellite, concatemer
• CPC Class Codes: C12Q 2525/191 incorporating an adaptor
• CPC Class Codes: C12Q 2531/125 Rolling circle
• CPC Class Codes: C12Q 2533/107 Probe or oligonucleotide ligation
• CPC Class Codes: C12Q 2565/518 characterised by the immobilisation of the nucleic acid sample or target
• CPC Class Codes: Y10T 436/143333 Saccharide [e.g., DNA, etc.]

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 determining the identification of a first nucleotide at a detection position of a target sequence comprising a plurality of detection positions, said method comprising:

(a) providing a plurality of concatemers, wherein each concatemer comprises a plurality of monomers and each monomer comprises;

i) a first target domain of said target sequence comprising a first set of target detection positions;

ii) a first adaptor comprising a Type IIs endonuclease restriction site;

iii) a second target domain of said target sequence comprising a second set of target detection positions; and

iv) a second interspersed adaptor comprising a Type IIs endonuclease restriction site;

(b) identifying said first nucleotide.

2. A method according to claim 1 wherein said target sequence concatemers are immobilized on a surface.

3. A method according to claim 2 wherein said identifying step comprises:

(a) contacting said concatemers with a set of sequencing probes each comprising;

i) a first domain complementary to one of said adaptors;

ii) a unique nucleotide at a first interrogation position; and

iii) a label;

under conditions wherein if said unique nucleotide is complementary to said first nucleotide, a sequencing probe hybridizes to said concatemer; and

(b) identifying said first nucleotide.

4. A method according to claim 2 wherein each adaptor comprises an anchor probe hybridization site, and said identifying step comprises:

(a) hybridizing anchor probes to said anchor probe hybridization sites;

(b) hybridizing sequencing probes to target detection positions adjacent to said adaptors;

(c) ligating adjacent hybridized sequencing and anchor probes to form ligated probes; and

(d) detecting said ligated probes to identify said first nucleotide.

5. A method according to claim 2 wherein each adaptor comprises an anchor probe hybridization site, and said identifying step comprises:

(a) hybridizing anchor probes to said anchor probe hybridization sites;

(b) adding a polymerase and at least one dNTP comprising a label, under conditions whereby if said dNTP is perfectly complementary to a detection position, said dNTP is added to the anchor probe to form an extended probe, thereby creating an interrogation position of the extended probe; and

(c) determining the nucleotide at the interrogation position of the extended probe.

6. A method according to claim 2 wherein a nucleotide at a second detection position is identified.

7. A method according to claim 6 wherein a nucleotide at a third detection position is identified.

8. A method according to claim 7 wherein a nucleotide at a fourth detection position is identified.

9. A method according to claim 8 wherein a nucleotide at a fifth detection position is identified.

10. A method according to claim 9 wherein a nucleotide at a sixth detection position is identified.

11. A method according to claim 2 wherein said surface is functionalized.

12. A method according to claim 11 wherein said functionalized surface comprises functional moieties selected from the group consisting of amines, silanes, and hydroxyls.

13. A method according to claim 2 wherein said surface comprises a plurality of spatially distinct regions comprising said immobilized concatemers.

14. A method according to claim 2 wherein said concatemers are immobilized on said surface using capture probes.

15. A method according to claim 1 further comprising fragmenting genomic nucleic acid to form target sequences.

16. A method according to claim 1 wherein the Type IIs endonuclease restriction sites of said first and second adaptors are the same.

17. A method according to claim 1 wherein the Type IIs endonuclease restriction sites of said first and second adaptors are different.

18. A substrate comprising a plurality of immobilized concatemers, each monomer of said concatemer comprising:

a) a first target sequence;

b) a first adaptor comprising a Type IIs endonuclease restriction site;

c) a second target sequence; and

d) a second interspersed adaptor comprising a Type IIs endonuclease restriction site.

19. A substrate according to claim 18 wherein each monomer further comprises a third target sequence and a third interspersed adaptor comprising a Type IIs endonuclease restriction site.

20. A substrate according to claim 19 each monomer further comprises a fourth target sequence and a fourth interspersed adaptor comprising a Type IIs endonuclease restriction site.

21. A substrate according to claim 18 wherein said substrate is glass.

22. A substrate according to claim 21 wherein said glass is functionalized.

23. A substrate according to claim 18 wherein said substrate comprises capture probes and said concatemers are immobilized by hybridization to said capture probes.

24. A substrate according to claim 18 wherein the Type IIs endonuclease restriction sites of said first and second adaptors are the same.

25. A substrate according to claim 18 wherein the Type IIs endonuclease restriction sites of said first and second adaptors are different.

26. A substrate according to claim 18 wherein said target sequences are genomic nucleic acid sequences.

27. A substrate according to claim 26 wherein said genomic nucleic acid sequences are human.

28. A method of inserting multiple adaptors in a target sequence comprising:

(a) ligating a first adaptor to one terminus of said target sequence, wherein the adaptor comprises a binding site for a restriction enzyme;

(b) circularizing the product from step (i) to create a first circular polynucleotide;

(c) cleaving the circular polynucleotide with a restriction enzyme, wherein the restriction enzyme is able to bind to the binding site within the first adaptor;

(d) ligating a second adaptor, wherein said second adaptor comprises a binding site for a restriction enzyme;

(e) circularizing the product from step (d) to create a second circular polynucleotide;

wherein steps (c) through (e) are optionally repeated to insert a desired number of adaptors in the target sequence.

29. A method according to claim 28 wherein said binding site of said first adaptor comprises a Type IIs endonuclease restriction site.

30. A method according to claim 28 wherein said binding site of said second adaptor comprises a Type IIs endonuclease restriction site.

31. A method according to claim 28 wherein said circularization step comprises adding a CircLigase™

enzyme.

32. A method according to claim 28 wherein said circularization step comprises:

(a) adding a circularization sequence to a second terminus of said target sequence;

(b) hybridizing a bridge template to at least a portion of said adaptor and a portion of said circularization sequence;

(c) ligating said first and second termini together to circularize the target sequence.

33. A method for identifying a nucleotide sequence of a target sequence, the method comprising the steps of:

(a) providing a plurality of interspersed adaptors within a target sequence, each interspersed adaptor having at least one boundary with the target sequence; and

(b) determining the identity of at least one nucleotide adjacent to at least one boundary of at least two interspersed adaptors, thereby identifying a nucleotide sequence of the target sequence.

34. A library of polynucleotides comprising more than one nucleic acid fragment, each fragment comprising a plurality of interspersed adaptor, wherein each interspersed adaptor has t least one end having different non-cross-hybridizable sequence with respect to the sequences of every other interspersed adaptor of the plurality.

35. The library of claim 34, wherein the plurality of interspersed adaptors is in a predetermined order.

36. The library of claim 35, wherein the predetermined order of the interspersed adaptors is the same for every nucleic acid fragment.

37. The library of claim 34, wherein each of said nucleic acid fragments is a closed single stranded DNA circle.

38. A method of identifying a nucleotide sequence of a target sequence, the method comprising the steps of:

(a) providing an amplicon from each of a plurality of fragments of the target sequence, each fragment containing a plurality of interspersed adaptors at predetermined sites, and each amplicon comprising multiple copies of a fragment and the amplicons including a number of fragments that substantially covers the target sequence;

(b) providing a random array of amplicons fixed to a surface at a density such that at least a majority of the amplicons are optically resolvable;

(c) hybridizing one or more sequencing probes to the random array under conditions that permit the formation of perfectly matched duplexes between the one or more sequencing probes and complementary sequences on the interspersed adaptors,

(d) identifying at least one nucleotide adjacent to at least one interspersed adaptor by extending the one or more sequencing probes in a sequence specific reaction; and

(e) repeating steps (c) and (d) until a nucleotide sequence of the target sequence is identified.

39. A method of identifying a nucleotide sequence of a target sequence, the method comprising the steps of:

(a) providing a random array of concatemers fixed to a planar surface, wherein said surface has an array of optically resolvable discrete spaced apart regions, and wherein each discrete spaced apart region has an area of less than 1 μ

m² and substantially all such regions have at most one of said concatemers attached, each concatemer comprising multiple copies of a fragment of the target sequence, each such fragment continuing a plurality of interspersed adaptors at predetermined sites, and the number of different concatemers such that their respective fragments substantially cover the target polynucleotide;

(b) hybridizing one or more probes from a first set of probes to the random array under conditions that permit the formation of perfectly matched duplexes between the one or more probes and complementary sequences on the concatemers;

(c) hybridizing one or more probes from a second set of probes to the random array under conditions that permit the formation of perfectly matched duplexes between the one or more probes and complementary sequences on the concatemers;

(d) ligating probes from the first and second sets which are hybridized to a concatemer at contiguous sites;

(e) identifying the sequences of the ligated probes; and

(f) repeating steps (b) through (e) to identify the nucleotide sequence of the target sequence.

40. A method of identifying a nucleotide sequence of a target sequence, the method comprising the steps of:

(a) providing a plurality of concatemers from the target sequence, each concatemer comprising multiple copies of a fragment of the target sequence, each fragment containing a plurality of interspersed adaptors at predetermined sites;

(b) providing a random array of concatemers fixed to a surface at a density such that at least a majority of the concatemers are optically resolvable;

(c) identifying a sequence of at least a portion of each fragment adjacent to at least one interspersed adaptor in at least one concatemer, thereby identifying a nucleotide sequence of the target sequence.

41. The method of claim 40, wherein said plurality of concatemers includes a number of fragments such that said fragments substantially cover said target sequence.

42. The method of claim 41 further comprising a step of reconstructing a nucleotide sequence of the target sequence from the identities of the sequences of said portions of said fragments of said concatemers.