ALTERNATIVE NUCLEIC ACID SEQUENCING METHODS

• US 20090062132A1
• Filed: 08/28/2008
• Published: 03/05/2009
• Est. Priority Date: 08/29/2007
• Status: Abandoned Application

• Current Assignee: Applied Biosystems LLC (Thermo Fisher Scientific Incorporated)
• Original Assignee: Applied Biosystems LLC (Thermo Fisher Scientific Incorporated)
• Inventors: Borner, Scott R.
• Application Number:
  US12/200,595
• Publication Number:
  US 20090062132A1
• Time in Patent Office: Days
• US Class Current: 506/7
• 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 2533/107 Probe or oligonucleotide ligation
• CPC Class Codes: C12Q 2535/101 Sanger sequencing method, i.e. oligonucleotide sequencing using primer elongation and dideoxynucleotides as chain terminators
• CPC Class Codes: C12Q 2537/149 Sequential reactions
• CPC Class Codes: C12Q 2565/301 Pyrophosphate (PPi)
• CPC Class Codes: C12Q 2565/627 being a mass spectrometer
• CPC Class Codes: C40B 20/08 Direct analysis of the library members per se by physical methods, e.g. spectroscopy
• CPC Class Codes: G16B 30/00 ICT specially adapted for sequence analysis involving nucleotides or amino acids
• CPC Class Codes: G16B 30/20 Sequence assembly

Abstract

Embodiments are provided that provide for parallel sequencing of nucleic acid segments. In some embodiments, a single sequence is sequenced by at least two different sequencing techniques and the results compared, allowing for deficiencies or strengths of one technique to be complemented by the second technique.

Claims

21. A method of sequencing a polynucleotide of interest, said method comprising:

applying a first nucleic acid sequencing chemistry to a clonal library derived from a polynucleotide of interest;

determining a sequence of a first region of the polynucleotide using a first set of nucleic acid sequencing reagents; and

determining a sequence of a second region of the polynucleotide using a second set of nucleic acid sequencing reagents, wherein the first set of nucleic acid sequencing reagents are different than the second set of nucleic acid sequencing reagents.

1. A method for sequencing a polynucleotide, said method comprising:

determining a sequence of a first region of a polynucleotide using a first set of nucleic acid sequencing reagents; and

determining a sequence of a second region of the polynucleotide using a second set of nucleic acid sequencing reagents, wherein the first set of nucleic acid sequencing reagents are different from the second set of nucleic acid sequencing reagents.

2. The method of claim 1, wherein the first region and the second region overlap.

3. The method of claim 1, wherein the first region and the second region are on different strands of the polynucleotide.

4. The method of claim 3, wherein the first region and the second region overlap by at least 5 nucleotides.

5. The method of claim 4, wherein the first and second region overlap by at least 10 nucleotides.

6. The method of claim 5, wherein the first region can be adjacent to the second region.

7. The method of claim 1, wherein the polynucleotide is immobilized on a solid support.

8. The method of claim 7, wherein the solid support can be a bead.

9. The method of claim 1, wherein the polynucleotide can be a single molecule.

10. The method of claim 1, wherein the polynucleotide can be an amplified clone.

11. The method of claim 10, wherein the amplified clone is produced by PCR.

12. The method of claim 11, wherein the PCR is emulsion PCR.

13. The method of claim 10, wherein the amplification takes place in a semisolid support.

14. The method of claim 10, wherein the amplification takes place on a solid support.

15. The method of claim 10, wherein the amplified clone is present on an array.

16. The method of claim 1, wherein the first sequencing chemistry or the second sequencing chemistry can be a sequencing by ligation chemistry.

17. The method of claim 1, wherein the first sequencing chemistry or the second sequencing chemistry can be a reversible terminator chemistry.

18. The method of claim 1, wherein the first sequencing chemistry or the second sequencing chemistry can be a pyrosequencing chemistry

19. The method of claim 16, wherein the first sequencing chemistry and the second sequencing chemistry can be are sequencing by ligation chemistry.

20. The method of claim 17, wherein the first sequencing chemistry and the second sequencing chemistry are reversible terminator chemistry.

22. The method of claim 21, wherein the clonal library comprised the genome of an organism of interest.

23. The method of claim 22, wherein the organism of interest is prokaryotic.

24. The method of claims 22, wherein the organism of interest is eukaryotic.

25. The method of claim 22, wherein the clonal library comprises clones derived from amplicons derived from a genome of interest.

26. The method of claim 21, wherein the clonal library is derived from a nucleic acid library

27. A method of sequencing a polynucleotide, said method comprising:

determining a sequence of a first region of the polynucleotide using a first set of nucleic acid sequencing reagents, whereby a first nucleic acid sequence is produced;

determining a sequence of a second region of the polynucleotide using a second set of nucleic acid sequencing reagents, wherein the first set of nucleic acid sequencing reagents are different than the second set of nucleic acid sequencing reagents, whereby a second nucleic acid sequence is produced, wherein the first region and the second region have at least 1 nucleotide base position in common; and

comparing the first nucleic acid sequence and the second nucleic acid.

28. A system for determining a base sequence of a polynucleotide of interest, the system comprising:

an array of polynucleotides for analysis;

a flow cell containing the array and having at least one input port;

a reservoir set comprising a first set of nucleic acid sequencing reagents, wherein the reservoir set is connection with an input port;

a reservoir set comprising a second set of nucleic acid sequencing reagents, wherein the reservoir set is in fluid connection with an input port;

a first optical signal collector configured to detect optical signal generated by reactions between the first set of nucleic acid sequencing reagents and the polynucleotides for analysis; and

a second optical signal collector configured to detect optical signal generated by reactions between the second set of nucleic acid sequencing reagents and the polynucleotides for analysis.

29. The system of claim 26, wherein the system further comprises a laser configured to induce excitation of fluorescent signal present on the array of polynucleotides.

27-1. The system of claim 26, wherein the first optical signal collector and the second optical signal collector are the same component.

28-2. The system of claim 26, wherein at least one of the optical signal collectors comprises a CCD.

30. A method for sequencing a polynucleotide, said method comprising:

fragmenting a polynucleotide for analysis into a plurality of polynucleotide fragments;

clonally amplifying at least a part of the polynucleotide fragments, whereby a set of fragment clones is produced;

sequencing a first portion of the set of fragment clones, with a first set of nucleic acid sequencing reagents, whereby a first nucleotide base sequence assembly is produced;

producing error values for at least some of the bases in the second nucleotide base sequence assembly;

sequencing a second portion of the set of fragment clones, with a second set of nucleic acid sequencing reagents, whereby a second nucleotide base sequence assembly is produced;

producing error values for at least some of the bases in the second nucleotide base sequence assembly;

comparing the first nucleotide base sequence assembly with the second nucleotide base sequence assembly; and

selecting at least one base identity between the first and second base sequence assemblies based upon a lower error value for the base identity in the corresponding nucleotide base sequence assembly compared to the base identity of the base in the other base sequence assembly.