Efficient shotgun sequencing methods

• US 8,298,768 B2
• Filed: 12/01/2008
• Issued: 10/30/2012
• 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
• Primary Examiner(s): Horlick, Kenneth R.
• Assistant Examiner(s): Tung, Joyce
• Application Number:
  US12/325,922
• Publication Number:
  US 20090318304A1
• Time in Patent Office: 1,429 Days
• Field of Search: 435/6.12, 435/91.2, 435/91.52
• US Class Current: 435/6.12
• 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 determining sequence of 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, wherein each of said plurality of oligonucleotides corresponds to at least one of the sequences identified in step (b);

(d) selecting, from a library of fragments of the target nucleic acid, fragments that hybridize with the target-specific oligonucleotides synthesized in step (c);

(e) sequencing fragments selected in step (d) to produce sequence information for the selected fragments; and

(f) assembling sequence information for the selected fragments determined in step (e) with the primary sequence information determined in step (a) to produce an assembled sequence, thereby determining sequence of the target nucleic acid.

2. The method of claim 1, further comprising:

(g) identifying missing sequences and/or low confidence sequences in the target nucleic acid from the assembled sequence information;

(h) synthesizing a plurality of target-specific oligonucleotides, wherein each of said plurality of oligonucleotides corresponds to at least one of the sequences identified in step (g);

(i) selecting fragments from a library of fragments of the target nucleic acid that hybridize with the target-specific oligonucleotides synthesized in step (h);

(j) sequencing fragments selected in step (i) to produce sequence information for said fragments; and

then

(k) assembling sequence information determined in previous steps of the method; and

(l) optionally repeating steps (g) through (k).

3. The method of claim 2, which is a method for sequencing human genomic DNA.

4. The method of claim 1, wherein step (c) comprises synthesizing at least about 10,000 target-specific oligonucleotides.

5. The method of claim 1, wherein step (c) comprises synthesizing at least about 100,000 target-specific oligonucleotides.

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

7. The method of claim 1, wherein step (a) comprises sequencing a first library of fragments of the target nucleic acid, and step (d) comprises hybridizing said plurality of target-specific oligonucleotides to a second library of fragments of the target nucleic acid.

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

9. The method of claim 1, wherein each of said plurality of target-specific oligonucleotides is attached to a solid support.

10. The method of claim 9, wherein the target-specific oligonucleotides are attached to the same solid support.

11. The method of claim 9, wherein each of said target-specific oligonucleotides is attached to a different solid support.

12. The method of claim 1

wherein each of said target-specific oligonucleotides comprises a binding moiety for attachment to a solid support, and

wherein step (d) comprises hybridizing said target-specific oligonucleotides to said library of fragments;

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

13. The method of claim 1 wherein step (e) comprises amplifying the library of fragments, thereby producing an amplified library, then sequencing the amplified library .

14. The method of claim 13 comprising amplifying the library by circle-dependent amplification.

15. The method of claim 14 comprising amplifying the library by circle-dependent amplification using the target-specific oligonucleotides as primers.

16. The method of claim 1 wherein the fragments selected in step (d) comprise from about 0.1 percent to about 10 percent of sequence of the target nucleic acid.

17. The method of claim 1 wherein computerized input of sequence readings and computerized assembly of the sequence readings are used to produce the sequence information in step (a).

18. The method of claim 1, wherein the target-specific oligonucleotides are designed by computer.

19. The method of claim 18 wherein computerized design of the target-specific oligonucleotides is integrated with computerized input and assembly of the sequence readings.

20. The method of claim 1 wherein step (b) comprises identifying about 100to about 100,000 missing sequences and/or low confidence sequences.

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

22. 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).

23. 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).

24. 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.

25. The method of claim 1, wherein the library of fragments in step (c) is a library of DNA concatemers, each concatemer comprising multiple copies of a fragment of the target nucleic acid concatenated in a single DNA strand.

26. 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 target nucleic acid from the nucleotide sequence information obtained in step (a);

(c) enriching fragments of the target nucleic acid from a fragment library according to whether they correspond to a sequence of interest identified in step (b);

(d) obtaining nucleotide sequence information for the fragments enriched in step (c); and

(e) assembling nucleotide sequence information determined in step (e) and step (a).

27. The method of claim 26, further comprising:

(f) identifying missing sequences and/or low confidence sequences in the target nucleic acid from the assembled information;

(g) enriching fragments of the target nucleic acid from a fragment library according to whether they correspond to a sequence of interest identified in step (f);

(h) obtaining nucleotide sequence information for the fragments enriched in step (g); and

then

(i) assembling sequence information determined in previous steps of the method; and

(j) optionally repeating steps (f) through (i).

28. The method of claim 26, wherein step (b) comprises identifying a sequence that is missing from the primary sequence information obtained in step (a).

29. The method of claim 26, wherein step (b) comprises identifying a sequence that comprises low confidence sequences in the primary sequence information obtained in step (a).

30. The method of claim 26, wherein step (b) comprises identifying a sequence that comprises low confidence base calls in the primary sequence information obtained in step (a).

31. The method of claim 26, wherein step (b) comprises identifying a missing sequences or low confidence sequence reads by comparing the primary sequence information with a reference sequence.

32. An improved method for sequencing a human genome, wherein the method comprises preparing overlapping fragments of the genome, obtaining multiple sequence reads for said overlapping fragments of the genome;

and assembling the reads into assembled sequence information, the improvement comprising;

(a) assembling sequence reads from fragments of the genome to obtain a primary assembly;

(b) identifying missing sequences, low confidence sequences, and/or sequences that differ between the primary assembly and a reference sequence in said human genome from the primary assembly;

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

(d) selecting fragments from a library of fragments of the target nucleic acid that hybridize with the oligonucleotides synthesized in step (c);

(e) obtaining sequence reads for the fragments selected in step (d); and

(f) assembling sequence reads obtained in step (e) with the primary assembly, thereby obtaining more complete sequence information.

33. 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) assembling sequence reads from fragments of the genome to obtain a primary assembly;

(b) identifying missing sequences and/or low confidence sequences in said human genome from the primary assembly;

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

(d) selecting fragments from a library of fragments of the target nucleic acid that hybridize with the oligonucleotides synthesized in step (c);

(e) obtaining sequence reads for the fragments selected in step (d); and

(f) assembling sequence reads obtained in step (e) with the primary assembly, thereby obtaining more complete sequence information.

34. The apparatus of claim 33, configured and programmed for sequencing a genome of a human organism according to a method that comprises the following additional steps:

(g) identifying missing sequences, low confidence sequences, and/or sequences that differ between the primary assembly and a reference sequence in the target nucleic acid from the assembled sequence information;

(h) synthesizing a plurality of target-specific oligonucleotides, wherein each of said plurality of oligonucleotides corresponds to at least one of the sequences identified in step (g);

(i) selecting fragments from a library of fragments of the target nucleic acid that hybridize with the target-specific oligonucleotides synthesized in step (h);

(j) sequencing fragments selected in step (i) to produce sequence information for said fragments; and

then

(k) assembling sequence information determined in previous steps of the method; and

(l) optionally repeating steps (g) through (k).