Efficient Shotgun Sequencing Methods

• US 20090318304A1
• Filed: 12/01/2008
• Published: 12/24/2009
• Est. Priority Date: 11/29/2007
• Status: Active Grant

• Current Assignee: Complete Genomics Incorporated (BGI Genomics Co., Ltd.)
• Original Assignee: Complete Genomics Incorporated (BGI Genomics Co., Ltd.)
• Inventors: Drmanac, Radoje, Reid, Clifford
• Granted Patent: US 8,298,768 B2
• Time in Patent Office: Days
• US Class Current: 506/9
• 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 2537/149 Sequential reactions
• CPC Class Codes: C12Q 2549/119 using nested primers
• CPC Class Codes: C12Q 2565/137 Chromatographic separation

Abstract

Methods are provided for efficient shotgun sequencing to allow efficient selection and sequencing of nucleic acids of interest contained in a library. The nucleic acids of interest can be defined any time before or after preparation of the library. One example of nucleic acids of interest is missing or low confidence genome sequences resulting from an initial sequencing procedure. Other nucleic acids of interest include subsets of genomic DNA, RNA or cDNAs (exons, genes, gene sets, transciptomes). By designing an efficient (simple to implement, speedy, high specificity, low cost) selection procedure, a more complete sequence is achieved with less effort than by using highly redundant shotgun sequencing in an initial sequencing procedure

Claims

1. A method of sequencing a target nucleic acid comprising:

a. sequencing the target nucleic acid to produce a primary target nucleic acid sequence that comprises one or more sequences of interest;

b. synthesizing a plurality of target-specific oligonucleotides, wherein each of said plurality of oligonucleotides corresponds to at least one of said sequences of interest;

c. providing a library of fragments of the target nucleic acid that hybridize to said plurality of oligonucleotides;

d. sequencing the library of fragments to produce a secondary target nucleic acid sequence.

2. The method of claim 1 wherein the target nucleic acid is genomic DNA of an organism.

3. The method of claim 2 wherein the target nucleic acid is human genomic DNA.

4. The method of claim 1 comprising synthesizing at least about 10,000 target-specific oligonucleotides.

5. The method of claim 1 comprising synthesizing at least about 100,000 target-specific oligonucleotides.

6. The method of claim 1 wherein sequencing the target nucleic acid comprises sequencing a first shotgun library of fragments of the target nucleic acid that represents a majority of sequences of the target nucleic acid.

7. The method of claim 6 comprising hybridizing said plurality of target-specific oligonucleotides to a second shotgun library of fragments of the target nucleic acid.

8. The method of claim 7 wherein the second shotgun library is an aliquot of the first shotgun library.

9. The method of claim 1 comprising hybridizing a pool of said plurality of target-specific oligonucleotides to the library of fragments.

10. The method of claim 1 comprising providing a plurality of constructs that each comprise a fragment of the target nucleic acid and separating constructs that hybridize to said plurality of target-specific oligonucleotides from constructs that do not hybridize to said plurality of target-specific oligonucleotides, thereby producing the library.

11. The method of claim 10 wherein each of said plurality of target-specific oligonucleotides is attached to a solid support.

12. The method of claim 10 wherein each of said plurality of target-specific oligonucleotides is attached to a single solid support.

13. The method of claim 10 wherein said each of said plurality of target-specific oligonucleotides is attached to a different solid support.

14. The method of claim 1 wherein each of said plurality of target-specific oligonucleotides comprises a binding moiety for attachment of each of said plurality of target-specific oligonucleotides to a solid support, the method comprising:

providing a plurality of constructs that each comprise a fragment of the target nucleic acid;

hybridizing said plurality of target-specific oligonucleotides to said plurality of constructs; and

attaching each of said plurality of target-specific oligonucleotides to a solid support.

15. The method of claim 1 wherein sequencing the library of constructs comprises amplifying the library of constructs, thereby producing an amplified library of constructs, then sequencing the amplified library of constructs.

16. The method of claim 15 comprising amplifying the library of constructs by circle-dependent amplification.

17. The method of claim 16 comprising amplifying the library of constructs by circle-dependent amplification using the target-specific oligonucleotides as primers.

18. The method of claim 1 wherein the library of constructs comprises from about 0.1 percent to about 10 percent of sequence of the target nucleic acid.

19. The method of claim 1 wherein step (a) comprises providing a library of constructs, each construct comprising a fragment of the target nucleic acid, and sequencing said library of constructs, and step (c) comprises providing an aliquot of said library of constructs and sequencing said aliquot of said library of constructs.

20. The method of claim 1 comprising comparing the primary target nucleic acid sequence to a reference sequence, thereby identifying said one or more sequences of interest.

21. The method of claim 1 wherein said one or more sequences of interest are selected from a group consisting of a gap, a low confidence sequence, and a change of one or more nucleotides in target sequence as compared to a reference sequence.

22. The method of claim 1 wherein the secondary target nucleic acid sequence has a substantially higher redundancy than the primary target nucleic acid sequence.

23. The method of claim 22 wherein the secondary target nucleic acid sequence has a redundancy that is at least two-fold higher than the primary target nucleic acid sequence.

24. The method of claim 22 wherein the secondary target nucleic acid sequence has a redundancy that is at least four-fold higher than the primary target nucleic acid sequence.

25. The method of claim 1 wherein sequencing the target nucleic acid to produce a primary target nucleic acid sequence comprises computerized input of sequence readings and computerized assembly of the sequence readings to produce the primary target nucleic acid sequence.

26. The method of claim 25 comprising computerized design of the target-specific oligonucleotides.

27. The method of claim 26 wherein computerized synthesis of the target-specific oligonucleotides is integrated with the computerized input and assembly of the sequence readings and design of the target-specific oligonucleotides.

28. The method of claim 1 wherein said primary target nucleic acid sequence comprises at least about 100 sequences of interest to about 100,000 sequences of interest.

29. A method for sequencing genomic DNA comprising:

a. sequencing a first shotgun library of fragments of the genomic DNA to produce a primary genomic sequence that comprises one or more sequences of interest;

b. synthesizing a plurality of oligonucleotides, wherein each of said plurality of oligonucleotides corresponds to at least one of said sequences of interest;

c. hybridizing a pool of said plurality of oligonucleotides to a second shotgun library of fragments of the genomic DNA;

d. preparing an enriched library of fragments of said genomic DNA from the second shotgun library that hybridize to the pool of said plurality of oligonucleotides;

e. sequencing the enriched shotgun library to produce a secondary target nucleic acid sequence.

30. A method for sequencing a target nucleic acid comprising:

a. sequencing the target nucleic acid to produce a primary target nucleic acid sequence that comprises one or more sequences of interest;

b. synthesizing a plurality of target-specific oligonucleotides, wherein each of said plurality of oligonucleotides corresponds to at least one of said sequences of interest;

c. providing a library of fragments of the target nucleic acid;

d. hybridizing said plurality of target-specific oligonucleotides to the library of fragments of the target nucleic acid;

e. hybridizing universal oligonucleotides to the library of fragments of the target nucleic acid;

f. ligating the universal oligonucleotides to adjacently hybridized target specific oligonucleotides, thereby producing universal oligonucleotide;

target-specific oligonucleotide hybrids;

g. preparing an enriched library of fragments of the target nucleic acid by capturing fragments of the target nucleic acid that are hybridized to the universal oligonucleotide;

target-specific oligonucleotide hybrids

h. sequencing the enriched library to produce a secondary target nucleic acid sequence.

31. A method for sequencing a target nucleic acid comprising:

a. sequencing the target nucleic acid to produce a primary target nucleic acid sequence that comprises one or more sequences of interest;

b. synthesizing a plurality of target-specific oligonucleotides, wherein each of said plurality of oligonucleotides corresponds to at least one of said sequences of interest;

c. providing a library of constructs, each construct comprising a fragment of the target nucleic acid;

d. hybridizing said plurality of oligonucleotides to the library of constructs;

e. amplifying the library of constructs using said plurality of oligonucleotides as primers, thereby producing an amplified library;

f. sequencing the amplified library to produce a secondary target nucleic acid sequence.