Molecular redundant sequencing

• US 7,901,889 B2
• Filed: 07/25/2008
• Issued: 03/08/2011
• Est. Priority Date: 07/26/2007
• Status: Active Grant

• Current Assignee: Pacific Bioscience of California Incorporated (L'Oreal SA)
• Original Assignee: Pacific Bioscience of California Incorporated (L'Oreal SA)
• Inventors: Turner, Stephen, Christians, Fred
• Primary Examiner(s): WHISENANT, ETHAN C
• Application Number:
  US12/220,674
• Publication Number:
  US 20090029385A1
• Time in Patent Office: 956 Days
• Field of Search: 435/6, 536/23.1, 536/24.3
• US Class Current: 435/6.12
• CPC Class Codes: C12Q 1/6869 Methods for sequencing

Abstract

Methods, systems and compositions where a target nucleic acid includes a registration sequence disposed therein for identification of the number or relative position of determined sequence from the template sequence. Particularly preferred aspects include a registration sequence in a circular template nucleic acid sequence which is, in turn, used in sequence by incorporation processes that rely upon template dependent, polymerase mediated primer extension in the identification of the sequence of the template.

Claims

1. A method of identifying a sequence context of a determined sequence of a target nucleic acid, comprising:

providing a known exogenous registration sequence at a selected location in an individual target nucleic acid molecule;

determining at least a portion of a sequence of the target nucleic acid molecule including the registration sequence; and

identifying a sequence context of the portion of the sequence of the target nucleic acid molecule determined in the determining step from a relative position of the registration sequence in the portion of the sequence.

2. The method of claim 1, wherein the determining step comprises sequencing an identical sequence of nucleotides multiple times within the individual nucleic acid molecule, and wherein the registration sequence is provided at a selected location relative to the identical sequence of nucleotides.

3. The method of claim 1, wherein the individual target nucleic acid molecule comprises a circular nucleic acid molecule.

4. The method of claim 1, wherein the identical sequence of nucleotides is provided multiple times within the target nucleic acid molecule.

5. The method of claim 3, wherein the determining step comprises determining the sequence of the portion of the target nucleic acid molecule wherein the registration sequence is determined at least twice, and the step of identifying the sequence context comprises determining that the entire circular nucleic acid molecule has been sequenced at least once.

6. The method of claim 1, wherein the target nucleic acid molecule comprises a linear nucleic acid.

7. The method of claim 6, wherein the registration sequence is provided at a plurality of locations within the target nucleic acid molecule.

8. The method of claim 6, wherein the registration sequence is provided proximal to one end of the target nucleic acid molecule.

9. The method of claim 1, wherein the step of determining at least a portion of the sequence of the target nucleic acid molecule comprises determining the sequence by incorporation of bases during a polymerase mediated template dependent primer extension reaction.

10. The method of claim 9, wherein the step of determining at least a portion of the sequence of the target nucleic acid molecule comprises a real-time sequencing by incorporation process.

11. The method of claim 1, wherein the registration sequence comprises from at least 5 bases to about 100 bases.

12. The method of claim 1, wherein the registration sequence comprises from at least 5 bases to about 50 bases.

13. The method of claim 1, wherein the registration sequence comprises from about 10 bases to about 20 bases.

14. A method of sequencing a complete target nucleic acid, comprising:

providing a known exogenous registration sequence within the target nucleic acid sequence;

circularizing the target nucleic acid sequence; and

sequencing individual target nucleic acid sequence molecules using a sequencing process that is nondestructive to the target nucleic acid sequence until the registration sequence has been sequenced at least twice.

15. The method of claim 14, wherein the sequencing process comprises identifying bases upon incorporation in a template directed, polymerase mediated primer extension reaction, wherein the target nucleic acid sequence comprises the template.

16. A method of determining a nucleic acid sequence, comprising:

providing an individual template nucleic acid molecule, a portion of which has at least a first sequence of nucleotides and an exogenous registration sequence at a selected location relative to the first sequence of nucleotides;

sequencing the individual nucleic acid molecule such that the first sequence of nucleotides and registration sequence are sequenced multiple times;

aligning sequence information from the sequencing step based at least in part upon the registration sequence identified in the sequencing step; and

determining a consensus sequence from the aligning step to determine the nucleic acid sequence of the first sequence of nucleotides.

17. The method of claim 16, wherein the template nucleic acid molecule is a circular nucleic acid, and the step of sequencing the first sequence of nucleotides multiple times comprises sequencing the circular nucleic acid multiple times.

18. The method of claim 16, wherein the first sequence of nucleotides is present multiple times in the template nucleic acid molecule.

19. The method of claim 16, wherein the first sequence of nucleotides is sequenced at least 5 times.

20. The method of claim 16, wherein the first sequence of nucleotides is sequenced at least 10 times.

21. The method of claim 16, wherein the first sequence of nucleotides is sequenced at least 20 times.

22. The method of claim 16, wherein the aligning step is performed by a computer.