Multibase delivery for long reads in sequencing by synthesis protocols

• US 9,005,929 B2
• Filed: 03/07/2014
• Issued: 04/14/2015
• Est. Priority Date: 12/23/2008
• Status: Active Grant

• Current Assignee: Illumina Incorporated
• Original Assignee: Illumina Incorporated
• Inventors: Ronaghi, Mostafa, Eltoukhy, Helmy A.
• Primary Examiner(s): HORLICK, KENNETH R
• Application Number:
  US14/200,674
• Publication Number:
  US 20140255919A1
• Time in Patent Office: 403 Days
• Field of Search: 435/91.1
• US Class Current: 435/91.1
• CPC Class Codes: C12Q 1/6837 using probe arrays or probe chips C12Q1/6874 takes precedence
• 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 2535/122 Massive parallel sequencing
• CPC Class Codes: C12Q 2565/543 characterised by the use of two or more capture oligonucleotide primers in concert, e.g. bridge amplification

Abstract

A method for obtaining nucleic acid sequence information that can include steps of (a) providing a first sequencing reagent to a target nucleic acid, wherein the first sequencing reagent comprises at least two different nucleotide monomers, (b) detecting the incorporation of a nucleotide monomer present in the first sequencing reagent into a polynucleotide strand complementary to at least a portion of the target nucleic acid, (c) providing a second sequencing reagent to said target nucleic acid, wherein the second sequencing reagent comprises one or more nucleotide monomers, at least one of the one or more nucleotide monomers being different from the nucleotide monomers present in the first sequencing reagent, and wherein the second sequencing reagent is provided subsequent to providing the first sequencing reagent, and (d) detecting the incorporation of a nucleotide monomer present in the second sequencing reagent into the polynucleotide strand.

Claims

1. A method for obtaining nucleic acid sequence information, said method comprising the steps of:

(a) providing a first sequencing reagent to a target nucleic acid, wherein said first sequencing reagent comprises at least two different nucleotide monomers;

(b) detecting the incorporation of a nucleotide monomer present in said first sequencing reagent into a polynucleotide strand complementary to at least a portion of said target nucleic acid;

(c) providing a second sequencing reagent to said target nucleic acid, wherein said second sequencing reagent comprises one or more nucleotide monomers, at least one of said one or more nucleotide monomers being different from the nucleotide monomers present in said first sequencing reagent, and wherein said second sequencing reagent is provided subsequent to providing said first sequencing reagent;

(d) detecting the incorporation of a nucleotide monomer present in said second sequencing reagent into the polynucleotide strand; and

(e) repeating steps (a) through (d) for said target nucleic acid, thereby obtaining sequence information for at least a portion of said target nucleic acid.

4. The method of claim 1, wherein said first sequencing reagent comprises no more than two nucleotide monomers.

5. The method of claim 4, wherein said second sequencing reagent comprises two different nucleotide monomers, said two different nucleotide monomers both different from the two nucleotide monomers of the first sequencing reagent.

6. The method of claim 1, wherein said first sequencing reagent comprises no more than three nucleotide monomers.

7. The method of claim 6, wherein said second sequencing reagent comprises one nucleotide monomer, said one nucleotide monomer different from said nucleotide monomers of the first sequencing reagent.

8. The method of claim 1, wherein said first sequencing reagent comprises a nucleotide monomer comprising a reversibly terminating moiety.

9. The method of claim 8, further comprising cleaving said reversibly terminating moiety from said nucleotide monomer.

10. The method of claim 1, wherein said first sequencing reagent is provided simultaneously to a plurality of target nucleic acids.

11. The method of claim 10, wherein said plurality of target nucleic acids comprise target nucleic acids having different nucleotide sequences.

12. The method of claim 11, wherein the said first sequencing reagent is provided to a plurality of target nucleic acids on a surface of an array in parallel.

2. A method for obtaining nucleic acid sequence information, said method comprising the steps of:

(a) detecting the incorporation of a nucleotide monomer present in a first sequencing reagent into a polynucleotide strand complementary to at least a portion of a target nucleic acid, wherein said first sequencing reagent comprises at least two different nucleotide monomers;

(b) removing at least a portion of said first sequencing reagent; and

(c) detecting the incorporation of a nucleotide monomer present in a second sequencing reagent into the polynucleotide strand, wherein said second sequencing reagent comprises one or more nucleotide monomers, at least one of said one or more nucleotide monomers being different from the nucleotide monomers present in said first sequencing reagent; and

(d) repeating steps (a) through (c) for said target nucleic acid, whereby sequence information for at least a portion of said target nucleic acid is obtained.

13. The method of claim 2, wherein said first sequencing reagent comprises no more than two nucleotide monomers.

14. The method of claim 13, wherein said second sequencing reagent comprises two different nucleotide monomers, said two different nucleotide monomers both different from the two nucleotide monomers of the first sequencing reagent.

15. The method of claim 2, wherein said first sequencing reagent comprises no more than three nucleotide monomers.

16. The method of claim 15, wherein said second sequencing reagent comprises one nucleotide monomer, said one nucleotide monomer different from said nucleotide monomers of the first sequencing reagent.

17. The method of claim 2, wherein said first sequencing reagent comprises a nucleotide monomer comprising a reversibly terminating moiety.

18. The method of claim 17, further comprising cleaving said reversibly terminating moiety from said nucleotide monomer.

19. The method of claim 2, wherein said first sequencing reagent is provided simultaneously to a plurality of target nucleic acids.

20. The method of claim 19, wherein said plurality of target nucleic acids comprise target nucleic acids having different nucleotide sequences.

21. The method of claim 20, wherein the said first sequencing reagent is provided to a plurality of target nucleic acids on a surface of an array in parallel.

3. A method for obtaining nucleic acid sequence information, said method comprising the steps of:

(a) detecting the incorporation of a nucleotide monomer present in a first sequencing reagent into a first polynucleotide strand complementary to at least a portion of a target nucleic acid, wherein said first sequencing reagent comprises at least two different nucleotide monomers;

(b) detecting the incorporation of a nucleotide monomer present in a second sequencing reagent into the first polynucleotide strand, wherein said second sequencing reagent comprises one or more nucleotide monomers, at least one of said one or more nucleotide monomers being different from the nucleotide monomers present in said first sequencing reagent;

(c) removing said first polynucleotide strand; and

(d) detecting the incorporation of a nucleotide monomer present in a third sequencing reagent into a second polynucleotide strand complementary to at least a portion of said target nucleic acid, wherein said third sequencing reagent comprises two or more nucleotide monomers, wherein at least one of said two or more nucleotide monomers is different from the at least two different nucleotide monomers present in said first sequencing reagent and at least one of said two or more nucleotide monomers is different from the one or more different nucleotide monomers present in said second sequencing reagent; and

(e) repeating steps (a) through (d) for said target nucleic acid.

22. The method of claim 3, wherein said first sequencing reagent comprises no more than two nucleotide monomers.

23. The method of claim 22, wherein said second sequencing reagent comprises two different nucleotide monomers, said two different nucleotide monomers both different from the two nucleotide monomers of the first sequencing reagent.

24. The method of claim 23, wherein said third sequencing reagent comprises two nucleotide monomers, and wherein one of said two nucleotide monomers is different from the at least two nucleotide monomers present in the first sequencing reagent.

25. The method of claim 3, wherein said first sequencing reagent comprises no more than three nucleotide monomers.

26. The method of claim 25, wherein said second sequencing reagent comprises one nucleotide monomer, said one nucleotide monomer different from said nucleotide monomers of the first sequencing reagent.

27. The method of claim 26, wherein said third sequencing reagent comprises three nucleotide monomers, and wherein one of said three nucleotide monomers is different from the at least two nucleotide monomers.

28. The method of claim 3, wherein said first sequencing reagent comprises a nucleotide monomer comprising a reversibly terminating moiety.

29. The method of claim 28, further comprising cleaving said reversibly terminating moiety from said nucleotide monomer.

30. The method of claim 3, wherein said first sequencing reagent is provided simultaneously to a plurality of target nucleic acids.

31. The method of claim 30, wherein said plurality of target nucleic acids comprise target nucleic acids having different nucleotide sequences.

32. The method of claim 31, wherein the said first sequencing reagent is provided to a plurality of target nucleic acids on a surface of an array in parallel.