Molecular redundant sequencing

• US 8,535,882 B2
• Filed: 02/03/2011
• Issued: 09/17/2013
• 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: Christians, Fred, Turner, Stephen
• Primary Examiner(s): Whisenant, Ethan C
• Application Number:
  US13/020,634
• Publication Number:
  US 20110212436A1
• Time in Patent Office: 957 Days
• Field of Search: 435/6.1, 536/23.1, 536/24.3
• US Class Current: 435/6.1
• 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 obtaining a consensus sequence for an individual circular nucleic acid molecule, comprising:

sequencing a first sequence of nucleotides present in the individual circular nucleic acid molecule a plurality of times to obtain a plurality of base calls at each position of the first sequence; and

calling bases within the first sequence of nucleotides present in the individual circular nucleic acid based upon at least a 75% consensus among the plurality of base calls for each position of the first sequence of nucleotides present in the individual circular nucleic acid molecule.

2. The method of claim 1, comprising calling bases within the first sequence of nucleotides present in the individual circular nucleic acid molecule based upon at least an 80% consensus among the plurality of base calls for each position of the first sequence of nucleotides present in the individual circular nucleic acid molecule.

3. The method of claim 1, comprising calling bases, within the first sequence of nucleotides present in the individual circular nucleic acid molecule based upon at least an 90% consensus among the plurality of base calls for each position of the first sequence of nucleotides present in the individual circular nucleic acid molecule.

4. The method of claim 1, wherein the first sequence of nucleotides is sequenced at least 2 times.

5. The method of claim 1, wherein the first sequence of nucleotides is sequenced at least 5 times.

6. The method of claim 1, wherein the first sequence of nucleotides is sequenced at least 10 times.

7. The method of claim 1, wherein the first sequence of nucleotides is sequenced at least 15 times.

8. The method of claim 1, wherein the first sequence of nucleotides is sequenced at least 20 times.

9. The method of claim 1, wherein the first sequence of nucleotides is sequenced by detecting polymerase mediated nucleotide addition in a template directed primer extension reaction.

10. A method of obtaining a consensus sequence for an individual nucleic acid molecule, comprising:

providing an individual nucleic acid molecule comprising multiple copies of a first sequence of nucleotides;

sequencing the multiple copies of the first sequence of nucleotides present in the individual nucleic acid molecule to obtain a plurality of base calls for each position of the first sequence, wherein the plurality of base calls for a single position comprises base calls from at least three of the multiple copies of the first sequence of nucleotides; and

calling bases within the first sequence of nucleotides based upon at least a 60% consensus among the plurality of base calls for each position of the first sequence of nucleotides.

11. The method of claim 10, comprising calling bases within the first sequence of nucleotides based upon at least an 80% consensus among the plurality of base calls for each position of the first sequence of nucleotides.

12. The method of claim 10, comprising calling bases within the first sequence of nucleotides based upon at least a 90% consensus among the plurality of base calls for each position of the first sequence of nucleotides.

13. The method of claim 10, wherein the first sequence of nucleotides is sequenced at least two times by sequencing at least two copies of the first sequence of nucleotides.

14. The method of claim 10, wherein the first sequence of nucleotides is sequenced at least five times by sequencing at least five copies of the first sequence of nucleotides.

15. The method of claim 10, wherein the first sequence of nucleotides is sequenced at least 10 times by sequencing at least 10 copies of the first sequence of nucleotides.

16. The method of claim 10, wherein the first sequence of nucleotides comprises at least 100 bases.

17. The method of claim 10, wherein the first sequence of nucleotides is sequenced by detecting polymerase mediated nucleotide addition in a template directed primer extension reaction.

18. The method of claim 17, wherein the detecting occurs in real time during the nucleotide addition.

19. The method of claim 17, wherein the addition occurs in the presence of four differentially labeled nucleotide analogs.

20. The method of claim 10, wherein the individual nucleic acid molecule further comprises a registration sequence at the beginning of each copy of the first sequence of nucleotides.