COMPOSITIONS AND METHODS FOR NUCLEIC ACID SEQUENCING

• US 20090298075A1
• Filed: 03/27/2009
• Published: 12/03/2009
• Est. Priority Date: 03/28/2008
• Status: Active Grant

• Current Assignee: Pacific Bioscience of California Incorporated (L'Oreal SA)
• Original Assignee: Pacific Bioscience of California Incorporated (L'Oreal SA)
• Inventors: Otto, Geoff, Turner, Stephen, Heiner, Cheryl, Ma, Congcong, Travers, Kevin
• Granted Patent: US 8,153,375 B2
• Time in Patent Office: Days
• US Class Current: 435/6.12
• CPC Class Codes: C12Q 1/6806 Preparing nucleic acids for analysis, e.g. for polymerase chain reaction [PCR] assay C12Q1/6804 takes precedence
• CPC Class Codes: C12Q 1/6855 Ligating adaptors
• CPC Class Codes: C12Q 1/6869 Methods for sequencing
• CPC Class Codes: C12Q 1/689 for bacteria
• CPC Class Codes: C12Q 2525/155 incorporating/generating a new priming site
• CPC Class Codes: C12Q 2525/301 Hairpin oligonucleotides
• CPC Class Codes: C12Q 2525/307 Circular oligonucleotides
• CPC Class Codes: C12Q 2531/125 Rolling circle
• CPC Class Codes: C12Q 2535/122 Massive parallel sequencing
• CPC Class Codes: G16B 30/00 ICT specially adapted for sequence analysis involving nucleotides or amino acids
• CPC Class Codes: G16B 30/10 Sequence alignment; Homology search

Abstract

Compositions and methods for nucleic acid sequencing include template constructs that comprise double stranded portions in a partially or completely contiguous constructs, to provide for redundant sequence determination through one or both of sequencing sense and antisense strands, and iteratively sequencing the entire construct multiple times. Additional sequence components are also optionally included within such template constructs. Methods are also provided for the use and preparation of these constructs as well as sequencing compositions for their application.

Claims

1. A method of determining a consensus sequence of nucleotides in a template nucleic acid segment, comprising:

providing a sense and an antisense strand of the template nucleic acid segment in a contiguous nucleic acid molecule;

sequencing both the sense and antisense strands in a polymerase mediated, template dependent sequencing process; and

determining a consensus sequence of the target nucleic acid segment from a sequence of the sense and antisense strands.

2. The method of claim 1, wherein the sense and antisense strands are disposed in a partially contiguous nucleic acid segment.

3. The method of claim 2, wherein the sense and antisense strands are linked at at least a first end by a first linking oligonucleotide.

4. The method of claim 1, wherein the sense and antisense strands are disposed in a completely contiguous nucleic acid segment.

5. The method of claim 4, wherein the sense and antisense strands are linked at each end by first and second linking oligonucleotides, respectively.

6. The method of claim 1, wherein the step of sequencing both the sense and antisense strands comprises sequencing at least one of the sense and antisense strands more than once.

7. The method of claim 4, wherein the sequencing step comprises contacting the contiguous nucleic acid molecule with a primer sequence that is complementary to at least a portion of the first linking oligonucleotide.

8. The method of claim 3, wherein the step of sequencing the sense and antisense strands comprises sequencing the completely contiguous nucleic acid segment more than once.

9. The method of claim 4, wherein the step of sequencing the sense and antisense strands comprises sequencing the completely contiguous nucleic acid segment more than twice.

10. The method of claim 1, wherein the polymerase mediated, template dependent sequencing process comprises detecting incorporation of each nucleotide incorporated by the polymerase.

11. The method of claim 10, wherein the step of detecting incorporation comprises detecting incorporation of each nucleotide incorporated by an individual polymerase.

12. A method of sequencing a nucleic acid segment, comprising:

providing a template nucleic acid segment comprising;

first and second complementary nucleic acid strands;

a first connecting nucleic acid linking a 3′

end of the first complementary nucleic acid strand to a 5′

end of the second complementary nucleic acid strand; and

a second connecting nucleic acid linking a 5′

end of the first complementary nucleic acid strand to a 3′

end of the second complementary nucleic acid strand;

contacting the template nucleic acid segment with a primer sequence complementary to at least a portion of the template nucleic acid segment; and

monitoring a template directed, polymerase mediated nucleic acid sequencing process to determine a sequence of nucleotides in at least one of the first and second complementary nucleic acid strands of the template nucleic acid segment.

13. The method of claim 12, wherein the sequencing process sequences both the first and second complementary nucleic acid strands of the template nucleic acid segment.

14. The method of claim 13, comprising the step of comparing sequence data from each of the first and second complementary nucleic acid strands of the template nucleic acid segment to determine a consensus sequence for the template nucleic acid segment.

15. The method of claim 12, wherein the sequencing process sequences at least one of the first and second complementary nucleic acid strands of the template nucleic acid segment more than once.

16. The method of claim 15, comprising comparing sequence data obtained from sequencing the at least one of the first and second complementary nucleic acid strands more than once, to determine a consensus nucleotide sequence of the template nucleic acid segment.

17. The method of claim 13, wherein the sequencing process sequences both the first and second complementary nucleic acid strands of the template nucleic acid segment more than once.

18. The method of claim 17, wherein at least one of the first connecting nucleic acid and the second connecting nucleic acid is a hairpin oligonucleotide.

27. The method of claim 12, further comprising a method for preparing the template nucleic acid segment, comprising:

providing the first and second complementary nucleic acid strands bound together in a double stranded nucleic acid;

linking the first connecting nucleic acid to the 3′

end of the first complementary nucleic acid strand and the 5′

end of the second complementary nucleic acid strand;

linking the second connecting nucleic acid to the 3′

end of the second complementary nucleic acid strand and the 5′

end of the first complementary nucleic acid strand; and

providing a primer recognition sequence within at least one of the first and second connecting nucleic acids.

19-26. -26. (canceled)

28-33. -33. (canceled)

34. A method of sequencing nucleic acid samples, comprising:

preparing template nucleic acid segments from each of a plurality of discrete nucleic acid samples, wherein the template nucleic acid segments comprise double stranded segments of the nucleic acid samples, a first strand of the double stranded segment being linked to a second strand of the double stranded segment by a linking oligonucleotide, wherein the linking oligonucleotide in each discrete nucleic acid sample comprises a unique, identifiable sequence characteristic;

pooling the template nucleic acid segments from the plurality of discrete nucleic acid samples;

sequencing the template nucleic acid segments pooled in the pooling step; and

identifying nucleic acid sequences as deriving from a discrete nucleic acid sample based at least in part on the unique identifiable sequence characteristic identified in the sequencing step.

35. A composition, comprising:

a template nucleic acid, comprising;

a double stranded nucleic acid segment comprising a first strand segment and a second strand segment substantially complementary to the first strand segment; and

at least a first linking oligonucleotide segment joining a 3′

end of the first strand segment to the 5′

end of the second strand segment;

a primer sequence capable of hybridizing to at least a portion of the template nucleic acid and initiating polymerase mediated nucleic acid synthesis; and

a polymerase enzyme.

36-45. -45. (canceled)

46. A template preparation kit, comprising:

a first linking oligonucleotide;

a primer sequence complementary to at least a portion of the first linking oligonucleotide; and

one or more ligation reagents for coupling the first linking oligonucleotide to a 3′

end of a first strand of a double stranded template and a 5′

end of a second strand of the double stranded template nucleic acid.

47. A system for sequencing a nucleic acid template, comprising:

a reaction mixture comprising;

a complex comprising a template nucleic acid, a polymerase enzyme and a primer sequence complementary to at least a portion of the template sequence, wherein the template sequence comprises a double stranded segment having a sense strand and an antisense strand, and at least a first linking oligonucleotide linking a 5′

end of the sense strand to the 3′

end of the antisense strand;

at least a first nucleotide or nucleotide analog bearing a detectable labeling group; and

a detection system configured to detect incorporation of the first nucleotide or nucleotide analog into a primer extension product by the polymerase.

48. The system of claim 47, further comprising a processor operably connected to the detection system, the processor including machine implemented instruction set for transforming signal data transmitted from the detection system to the processor to nucleic acid sequence information interpretable by a user.