Efficient shotgun sequencing methods

• US 9,238,834 B2
• Filed: 10/08/2012
• Issued: 01/19/2016
• 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 T., Reid, Clifford
• Primary Examiner(s): Horlick, Kenneth R.
• Assistant Examiner(s): Tung, Joyce
• Application Number:
  US13/647,338
• Publication Number:
  US 20130059737A1
• Time in Patent Office: 1,198 Days
• Field of Search: 435/6.12, 435/91.2, 435/91.52
• US Class Current: 1/1
• 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 for sequencing a target nucleic acid comprising:

(a) sequencing the target nucleic acid to produce primary sequence information for the target nucleic acid;

(b) identifying missing sequences and/or low confidence sequences in the target nucleic acid from the primary sequence information determined in step (a);

(c) synthesizing a plurality of target-specific oligonucleotides,

each corresponding to a sequence identified in step (b);

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

(e) hybridizing universal oligonucleotides to the library of fragments;

(f) ligating the universal oligonucleotides that have hybridized to the target nucleic acids to adjacently hybridized target-specific oligonucleotides, thereby producing universal oligonucleotide;

target-specific oligonucleotide ligation products; and

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

target-specific oligonucleotide ligation products.

2. The method of claim 1, further comprising:

(h) sequencing fragments from the enriched library prepared in step (g); and

(i) assembling sequence information from the fragments sequenced in step (h) with the primary sequence information determined in step (a) to produce an assembled sequence.

3. The method of claim 2, further comprising:

(j) identifying missing sequences and/or low confidence sequences in the target nucleic acid from the sequence assembled in step (i);

(k) synthesizing a plurality of target-specific oligonucleotides, each corresponding to a sequence identified in step (j);

(l) performing steps (d) to (i) using the target-specific oligonucleotides prepared in step (k);

(m) optionally repeating steps (j) to (l).

4. The method of claim 1, wherein the universal oligonucleotides comprise 5 to 15 consecutive universal bases.

5. The method of claim 4, wherein the universal oligonucleotides further comprise 1 to 5 sequence-specific bases.

6. The method of claim 1, wherein the universal oligonucleotides comprise 5 to 15 consecutive degenerate bases.

7. The method of claim 6, wherein the universal oligonucleotides further comprise 1 to 5 sequence-specific bases.

8. The method of claim 1, wherein the universal oligonucleotides comprise a capture tag.

9. The method of claim 8, wherein the capture tag is biotin.

10. The method of claim 8, wherein the universal oligonucleotide is phosphorylated at the 5′

end.

11. The method of claim 1, wherein the universal oligonucleotides comprise a formula 5′

-B_x—

N_y-Tag-3′

, where B denotes a sequence specific base, x is 1 to 5, N denotes a degenerate or universal base, y is 5 to 15, and Tag is a capture tag.

12. The method of claim 1, wherein the target specific oligonucleotides are 20 to 30 bases in length.

13. The method of claim 1, wherein the hybridizing and ligating are conducted under conditions such that universal oligonucleotides not ligated to target-specific oligonucleotides melt easily from the library constructs whereas the target-specific selection oligonucleotides do not.

14. The method of claim 1, wherein step (b) comprises identifying a sequence that is missing from the primary sequence information obtained in step (a).

15. The method of claim 1, wherein step (b) comprises identifying a sequence that comprises low confidence sequences in the primary sequence information obtained in step (a).

16. The method of claim 1, wherein step (b) comprises identifying a sequence that comprises low confidence base calls in the primary sequence information obtained in step (a).

17. The method of claim 1, wherein step (b) comprises identifying missing sequences, or low confidence sequence reads by comparing the primary sequence information with a reference sequence.

18. The method of claim 1, wherein the library of fragments in step (c) is a library of DNA amplicons, each amplicon comprising multiple copies of a fragment of the target nucleic acid.

19. A method for sequencing a target nucleic acid, comprising:

(a) obtaining nucleotide sequence information for at least a portion of the target nucleic acid;

(b) identifying missing sequences and/or low confidence sequences in the nucleotide sequence information;

(c) synthesizing a plurality of target-specific oligonucleotides, each corresponding to a sequence identified in step (b);

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

(e) hybridizing universal oligonucleotides to the library of fragments;

(f) ligating the universal oligonucleotides that have hybridized to the target nucleic acids to adjacently hybridized target-specific oligonucleotides, thereby producing universal oligonucleotide;

target-specific oligonucleotide ligation products;

(g) preparing an enriched library of fragments of the target nucleic acid by selecting fragments of the target nucleic acid from the library that are hybridized to the universal oligonucleotide;

target-specific oligonucleotide ligation products;

(h) sequencing fragments in the enriched library; and

(i) assembling sequence information determined in step (h) with the sequence information obtained in step (a), thereby obtaining more complete sequence information.

20. The method of claim 19, further comprising:

(j) identifying missing sequences and/or low confidence sequences in the target nucleic acid from the sequence assembled in step (i);

(k) synthesizing a plurality of target-specific oligonucleotides, each corresponding to a sequence identified in step (j);

(l) performing steps (d) to (i) using the target-specific oligonucleotides prepared in step (k);

(m) optionally repeating steps (j) to (l).

21. The method of claim 19, wherein the universal oligonucleotides comprise a plurality of universal and/or degenerate bases and 1 to 5 sequence-specific bases.

22. The method of claim 19, wherein the universal oligonucleotides comprise a capture tag.

23. The method of claim 19, wherein the hybridizing and ligating are conducted under conditions such that universal oligonucleotides not ligated to target-specific oligonucleotides melt easily from the library constructs whereas the target-specific selection oligonucleotides do not.

24. The method of claim 19, wherein the library of fragments in step (c) is a library of DNA amplicons, each amplicon comprising multiple copies of a fragment of the target nucleic acid.

25. A computer controlled apparatus configured and programmed for sequencing a genome of a human organism according to a method that comprises the following steps:

(a) obtaining nucleotide sequence information for at least a portion of the genome;

(b) identifying missing sequences and/or low confidence sequences in the nucleotide sequence information;

(c) synthesizing a plurality of target-specific oligonucleotides, each corresponding to a sequence identified in step (b);

(d) hybridizing said plurality of target-specific oligonucleotides to a library of fragments of the genome;

(e) hybridizing universal oligonucleotides to the library of fragments;

(f) ligating the universal oligonucleotides that have hybridized to the target nucleic acids to adjacently hybridized sequence-specific oligonucleotides, thereby producing universal oligonucleotide;

sequence-specific oligonucleotide ligation products;

(g) preparing an enriched library of fragments of the genome by capturing fragments of the genome nucleic acid from the library that are hybridized to the universal oligonucleotide;

sequence-specific oligonucleotide ligation products;

(h) sequencing fragments in the enriched library; and

(i) assembling sequence information determined in step (h) with the primary sequence information obtained from step (a).