Compositions and methods for nucleic acid sequencing

• US 8,455,193 B2
• Filed: 02/23/2012
• Issued: 06/04/2013
• 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: Travers, Kevin, Otto, Geoff, Turner, Stephen, Heiner, Cheryl, Ma, Congcong
• Primary Examiner(s): Lu, Frank
• Application Number:
  US13/403,789
• Publication Number:
  US 20120289408A1
• Time in Patent Office: 467 Days
• Field of Search: 435/6.1, 435/6.11, 435/6.12, 435/91.1, 435/91.2, 435/183, 436/94, 436/501, 536/23.1, 536/24.3, 536/24.33, 536/25.3
• US Class Current: 435/6.1
• 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 sequence of a double-stranded nucleic acid sample, comprising:

(a) locking the forward strand and the reverse strand of the nucleic acid sample together thereby forming a single-stranded circular pair-locked molecule, wherein the forward strand and the reverse strand are complementary to each other and both ends of the forward strand and both ends of the reverse strand are joined by linking oligonucleotides;

(b) performing single molecule sequencing of the circular pair-locked molecule thereby obtaining sequence data of the circular pair-locked molecule, wherein sequence data comprises sequences of the forward strand and the reverse strand of the circular pair-locked molecule; and

(c) determining the sequence of the double-stranded nucleic acid sample by comparing the sequences of the forward strand and the reverse strand of the circular pair-locked molecule.

2. The method of claim 1, wherein said locking the forward strand and the reverse strand of the nucleic acid sample together comprises joining 5′

end of the forward strand to 3′

end of the reverse strand and joining 3′

end of the forward strand to 5′

end of the reverse strand by said linking oligonucleotides, and wherein said linking oligonucleotides are two hairpin adaptors.

3. The method of claim 2, wherein said obtaining sequence data comprises annealing a primer complementary to at least part of at least one of the hairpin adaptors and extending the primer.

4. The method of claim 1, wherein said single molecule sequencing comprises sequencing by synthesis technology.

5. The method of claim 1, wherein said single molecule sequencing is performed in the presence of an electrochemical system comprising a nanopore sensor.

6. A method of determining a sequence of a double-stranded nucleic acid sample, comprising:

(a) linking the forward strand and the reverse strand of the nucleic acid sample together thereby forming a single-stranded circular template molecule, wherein the forward strand and the reverse strand are complementary to each other and both ends of the forward strand and both ends of the reverse strand are joined by linking oligonucleotides;

(b) performing a single molecule sequencing of the circular template molecule thereby obtaining sequence data of the circular template molecule, wherein sequence data comprises sequences of the forward strand and the reverse strand of the circular template molecule; and

(c) determining the sequence of the double-stranded nucleic acid sample by comparing the sequences of the forward strand and the reverse strand of the circular template molecule.

7. The method of claim 6, wherein the linking oligonucleotides are two hairpin adaptors and said linking the forward strand and the reverse strand of the nucleic acid sample together comprises joining 5′

end of the forward strand to 3′

end of the reverse strand and joining 3′

end of the forward strand to 5′

end of the reverse strand by said two hairpin adaptors.

8. The method of claim 7, wherein said obtaining sequence data comprises annealing a primer complementary to at least part of at least one of the hairpin adaptors and extending the primer.

9. The method of claim 6, wherein said single molecule sequencing comprises sequencing by synthesis technology.

10. The method of claim 6, wherein said single molecule sequencing is performed in the presence of an electrochemical system comprising a nanopore sensor.