Methods and Oligonucleotide Designs for Insertion of Multiple Adaptors Employing Selective Methylation

• US 20090203551A1
• Filed: 11/05/2008
• Published: 08/13/2009
• Est. Priority Date: 11/05/2007
• Status: Active Grant

• Current Assignee: Complete Genomics Incorporated (BGI Genomics Co., Ltd.)
• Original Assignee: Complete Genomics Incorporated (BGI Genomics Co., Ltd.)
• Inventors: DAHL, Fredrik, SPARKS, Andrew, DRMANAC, Radoje
• Granted Patent: US 7,901,890 B2
• Time in Patent Office: Days
• US Class Current: 506/26
• CPC Class Codes: C12Q 1/6855 Ligating adaptors
• CPC Class Codes: C12Q 1/6874 involving nucleic acid arrays, e.g. sequencing by hybridisation
• CPC Class Codes: C12Q 2521/125 Methyl transferase, i.e. methylase
• CPC Class Codes: C12Q 2521/313 Type II endonucleases, i.e. cutting outside recognition site
• CPC Class Codes: C12Q 2521/501 Ligase
• CPC Class Codes: C12Q 2521/531 Glycosylase
• CPC Class Codes: C12Q 2525/191 incorporating an adaptor
• CPC Class Codes: C12Q 2531/125 Rolling circle
• CPC Class Codes: C12Q 2537/163 blocking probe

Abstract

Aspects described and claimed herein provide methods to insert multiple DNA adaptors into a population of circular target DNAs at defined positions and orientations with respect to one another. The resulting multi-adaptor constructs are then used in massively-parallel nucleic acid sequencing techniques.

Claims

1. A method for selectively activating a recognition site for a Type IIs restriction endonuclease in a nucleic acid sequence, the method comprising:

(a) providing a nucleic acid sequence comprising first and second recognition sites for a Type IIs restriction endonuclease;

(b) amplifying the nucleic acid sequence using a uracil-containing primer that has a sequence that is complementary to the first recognition site, thereby producing an amplified nucleic acid sequence comprising a first recognition site for a Type IIs restriction endonuclease comprising, one or more uracils at or near the first recognition site, and a second recognition site for a Type IIs restriction endonuclease;

(c) degrading said one or more uracils at or near the first recognition site, thereby producing a single-stranded region in the first recognition site and protecting the first recognition site from methylation by a methylase that methylates unprotected recognition sites for the Type IIs restriction endonuclease;

(f) methylating the second recognition site with the methylase, inhibiting digestion of the nucleic acid sequence by the Type IIs restriction endonuclease resulting from recognition of the second recognition site; and

(g) making the single-stranded region double-stranded such that the Type IIs restriction endonuclease can recognize the first recognition site and digest the nucleic acid sequence.

2. A method of positioning a second adaptor with respect to a first adaptor in a nucleic acid template construct, said method comprising:

(a) providing a first linear construct, wherein said first linear construct comprises a target nucleic acid and a first adaptor, and wherein said first adaptor comprises a first recognition site for a first Type IIs restriction endonuclease;

(b) protecting said first recognition site from inactivation;

(c) inactivating unprotected restriction endonuclease recognition sites in said first linear construct;

(d) circularizing said first linear construct to form a first circular construct;

(e) applying said first Type IIs restriction endonuclease to said first circular construct to form a second linear construct, wherein said second linear construct comprises said first adaptor inserted within said target nucleic acid;

(f) ligating a second adaptor to said second linear construct to form said nucleic acid template construct, wherein said second adaptor comprises a second recognition site for a second Type IIs restriction endonuclease;

thereby positioning said second adaptor with respect to said first adaptor in said nucleic acid template construct.

3. The method of claim 2, wherein said protecting step (b) comprises rendering said first recognition site single-stranded.

4. The method of claim 3, wherein said rendering said first recognition site single-stranded comprises:

(a) amplifying said first linear construct with uracil-modified primer complementary to said first adaptor to produce first uracil-modified linear constructs; and

(b) degrading uracils in said first uracil-modified linear constructs, thereby rendering said first recognition site single-stranded.

5. The method of claim 2 further comprising:

(a) circularizing said second linear construct to form a second circular construct;

(b) protecting said second recognition site from inactivation;

(c) inactivating unprotected Type IIs restriction endonuclease recognition sites in said second circular construct;

(d) applying said second restriction endonuclease to said second circular construct to form a second linear construct, wherein said second linear construct comprises said first adaptor and said second adaptor inserted within said target nucleic acid;

(e) ligating a third adaptor to said second linear construct to form a third linear construct;

(f) circularizing said third linear construct, thereby forming said nucleic acid template construct.

6. The method of claim 5, further comprising repeating steps (h) through (1) to insert a desired number of further adaptors, wherein said protecting step is performed on each successively-added adaptor.

13. The method of claim 6, further comprising generating concatemers from said library of circular nucleic acid templates.

14. The method of claim 13, further comprising disposing said concatemers on a surface to form a random array.

15. The method of claim 14, further comprising identifying at least one nucleotide in at least one of said concatemers.

16. The method of claim 15, wherein said at least one nucleotide is adjacent to one of said first, second and third adaptors.

17. The method of claim 15, wherein said identifying comprises:

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

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

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

(iv) identifying the sequences of the ligated probes, thereby identifying said at least one nucleotide.

7. A method of making a library of circular nucleic acid templates each comprising a target nucleic acid sequence and at least two adaptors, said method comprising:

(a) providing fragments of genomic nucleic acid;

(b) adding a first arm of a first adaptor to one terminus of a plurality of said fragments;

(c) adding a second arm of a first adaptor to the other terminus of said plurality of said fragments to form first linear constructs, wherein said first and second arms of said first adaptor, when ligated, form said first adaptor and produce a first recognition site for a first Type IIs restriction endonuclease;

(d) protecting said first recognition site in said first linear constructs from inactivation;

(e) inactivating any unprotected first recognition sites present in said first linear constructs;

(f) circularizing said first linear constructs by ligating said first and second adaptor arms to form first circular constructs;

(g) cleaving said first circular constructs with said first Type IIs restriction endonuclease to form second linear constructs comprising said first adaptor inserted within said target nucleic acid, wherein said first Type IIs restriction endonuclease binds to said protected first recognition site and cleaves at a position in said first circular constructs outside of said first adaptor;

(h) adding a first arm of a second adaptor to one terminus of said plurality of said second linear constructs;

(i) adding a second arm of a second adaptor to the other terminus of said plurality of said fragments to form second linear constructs, wherein said first and second arms of said second adaptor, when ligated, form said second adaptor and form a second Type IIs recognition site;

(j) circularizing said second linear constructs by ligating said first and second adaptor arms of said second adaptor to form second circular constructs,

thereby making said library of circular nucleic acid templates.

8. The method of claim 7, wherein said fragments are generated by:

(a) isolating said genomic nucleic acid;

(b) fractionating said genomic nucleic acid;

(c) isolating fragments of a desired size; and

(d) modifying the termini of said fragments such that two fragment are unable to ligate to each other,

thereby generating said fragments.

9. The method of claim 8, wherein said isolating comprises using gel fractionation.

10. The method of claim 7, wherein said protecting step comprises:

(a) embedding uracils in said first recognition site;

(b) degrading said uracils.

11. The method of claim 10, wherein said inactivating step comprises applying a sequence-specific methylase that is only able to methylate double stranded sequences, wherein said sequence-specific methylase is specific for said first recognition site.

12. The method of claim 10, wherein said embedding comprises amplifying said first linear constructs with uracil-modified primers to produce first linear constructs with uracils embedded in said first recognition site.