SEQUENCING METHODS

• US 20100279882A1
• Filed: 04/30/2010
• Published: 11/04/2010
• Est. Priority Date: 05/01/2009
• Status: Abandoned Application

• Current Assignee: Illumina Incorporated
• Original Assignee: Illumina Incorporated
• Inventors: Betley, Jason Richard, Evers, Dirk, Ronaghi, Mostafa
• Application Number:
  US12/771,992
• Publication Number:
  US 20100279882A1
• Time in Patent Office: Days
• US Class Current: 506/8
• CPC Class Codes: C12Q 1/6869 Methods for sequencing
• CPC Class Codes: C12Q 2525/186 incorporating a non-extendable or blocking moiety
• CPC Class Codes: C12Q 2525/197 incorporating a spacer/coupling moiety
• 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

The present technology relates to molecular sciences, such as genomics. More particularly, the present technology relates to nucleic acid sequencing.

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 in the presence of a polymerase, said first sequencing reagent comprising one or more nucleotide monomers, wherein said one or more nucleotide monomers pair with no more than three nucleotide types in said target, thereby forming a polynucleotide complementary to at least a portion of said target; and

(b) providing a second sequencing reagent to said target nucleic acid, said second sequencing reagent comprising at least one nucleotide monomer, said at least one nucleotide monomer of said second sequencing reagent comprising a reversibly terminating moiety, wherein said second sequencing reagent is provided subsequent to providing said first sequencing reagent, whereby sequence information for at least a portion of said target nucleic acid is obtained.

2. The method of claim 1, further comprising identifying a homopolymer sequence of nucleotides in said target.

3. The method of claim 1, wherein said one or more nucleotide monomers pair with at least two nucleotide types in said target.

4. The method of claim 1, wherein said first sequencing regent comprises at least two different nucleotide monomers.

5. The method of claim 1, wherein said one or more nucleotide monomers lack a reversibly terminating moiety.

6. The method of claim 1 further comprising removing unincorporated second sequencing reagent.

7. The method of claim 6 further comprising removing said reversibly terminating moiety.

8. The method of claim 7 further comprising providing a third sequencing reagent comprising at least one nucleotide monomer comprising a reversibly terminating moiety.

9. The method of claim 7 further comprising removing unincorporated first sequencing reagent prior to removing said reversibly terminating moiety.

10. The method of claim 9 further comprising providing a third sequencing reagent comprising at least one nucleotide monomer comprising a reversibly terminating moiety.

11. The method of claim 9 further comprising repeating step (a) at least once prior to repeating step (b).

12. The method of claim 1, further comprising detecting incorporation of the at least one nucleotide monomer of said second sequencing reagent into said polynucleotide.

13. The method of claim 12, wherein said detecting comprises detecting a label.

14. The method of claim 12, wherein said detecting comprises detecting pyrophosphate.

15. The method of claim 12, wherein said at least one nucleotide monomer of said second sequencing reagent comprises a label.

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

17. The method of claim 16, wherein said plurality of target nucleic acids comprise target nucleic acids having different nucleotide sequences.

18. The method of claim 1, wherein said first sequencing reagent is provided in parallel to a plurality of target nucleic acids at separate features of an array.

19. The method of claim 18, wherein said plurality of target nucleic acids comprise target nucleic acids having different nucleotide sequences.

20. 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 in the presence of a polymerase, said first sequencing reagent comprising a plurality of different nucleotide monomers, wherein at least one nucleotide monomer of said plurality of nucleotide monomers comprises a reversibly terminating moiety, thereby forming a polynucleotide complementary to at least a portion of said target; and

(b) removing the reversibly terminating moiety of said at least one nucleotide monomer of said first sequencing reagent; and

(c) providing a second sequencing reagent to said target nucleic acid, said second sequencing reagent comprising at least one nucleotide monomer, said at least one nucleotide monomer of said second sequencing reagent comprising a reversibly terminating moiety, wherein said second sequencing reagent is provided subsequent to providing said first sequencing reagent, whereby sequence information for at least a portion of said target nucleic acid is obtained.

21. 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 in the presence of a ligase, wherein the first sequencing reagent comprises at least one oligonucleotide, wherein said oligonucleotide comprises a reversibly terminating moiety;

(b) removing the reversibly terminating moiety of said at least one oligonucleotide of said first sequencing reagent; and

(c) providing a second sequencing reagent to said target nucleic acid in the presence of a polymerase wherein said second sequencing reagent comprises at least one nucleotide monomer, wherein said nucleotide monomer comprises a reversibly terminating moiety, and wherein said second sequencing reagent is provided subsequent to providing said first sequencing reagent, whereby sequence information for at least a portion of said target nucleic acid is obtained.

22. 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 in the presence of a polymerase, said first sequencing reagent comprising one or more nucleotide monomers, wherein said one or more nucleotide monomers pair with no more than three nucleotide types in said target, thereby forming a polynucleotide complementary to at least a portion of said target; and

(b) providing a second sequencing reagent to said target nucleic acid, said second sequencing reagent comprising at least one nucleotide monomer, wherein said at least one nucleotide monomer pairs with no more than three nucleotide types in said target, wherein said second sequencing reagent is provided subsequent to providing said first sequencing reagent, and wherein a signal that indicates the incorporation of said at least one nucleotide monomer into the polynucleotide is generated, whereby sequence information for at least a portion of said target nucleic acid is obtained.

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

(a) providing a first low resolution sequence representation for a target nucleic acid, wherein said first low resolution sequence representation comprises an ordered series of determined regions and dark regions, wherein said determined regions comprise a sequence of at least two discrete nucleotides, wherein said dark regions are indicative of degenerate sequence composition, and wherein said dark regions intervene between said determined regions;

(b) providing a second low resolution sequence representation for said target nucleic acid, wherein said second low resolution sequence representation comprises an ordered series of determined regions and dark regions, wherein said determined regions comprise a sequence of at least two discrete nucleotides, wherein said dark regions are indicative of degenerate sequence composition, and wherein said dark regions intervene between said determined regions and wherein said sequence of at least two discrete nucleotides in said first low resolution sequence representation is different from said sequence of at least two discrete nucleotides in said second low resolution sequence representation; and

(c) comparing said first low resolution sequence representation and said second low resolution sequence representation to determine a sequence representation having a resolution higher than either the first low resolution sequences representation or second low resolution sequence representation alone.

24. The method of claim 23, wherein said sequence representation having a resolution higher than either the first low resolution sequences representation or second low resolution sequence representation comprises the sequence of said target nucleic acid at single nucleotide resolution.

25. The method of claim 23, wherein said dark regions are indicative of variable sequence length.

26. The method of claim 23, wherein said sequence of at least two discrete nucleotides in said first low resolution sequence representation is no longer than two nucleotides.

27. The method of claim 26, wherein said sequence of at least two discrete nucleotides in said second low resolution sequence representation is no longer than two nucleotides.

28. The method of claim 23, wherein said dark region in said first low resolution sequence representation is degenerate with respect to a pair of nucleotide types.

29. The method of claim 28, wherein said dark region in said second low resolution sequence representation is degenerate with respect to a pair of nucleotide types.

30. The method of claim 23, wherein said determined regions comprise a sequence of at least two discrete nucleotides from the target nucleic acid.

31. The method of claim 23, wherein said determined regions comprise a sequence of at least two discrete nucleotides that are complementary to nucleotides from the target nucleic acid.

32. A method for determining the presence of a target nucleic acid, said method comprising the steps of:

(a) providing a first low resolution sequence representation for a target nucleic acid, wherein said target nucleic acid is obtained from a first sample, wherein said first low resolution sequence representation comprises an ordered series of determined regions and dark regions, wherein said determined regions comprise a sequence of at least two discrete nucleotides, wherein said dark regions are indicative of degenerate sequence composition, and wherein said dark regions intervene between said determined regions;

(b) providing a second low resolution sequence representation for a second target nucleic acid, wherein said second target nucleic acid is obtained from a reference sample and has the sequence expected for the target nucleic acid, wherein said second low resolution sequence representation comprises an ordered series of determined regions and dark regions, wherein said determined regions comprise a sequence of at least two discrete nucleotides, wherein said dark regions are indicative of degenerate sequence composition, and wherein said dark regions intervene between said determined regions and wherein said sequence of at least two discrete nucleotides in said first low resolution sequence representation is different from said sequence of at least two discrete nucleotides in said second low resolution sequence representation; and

(c) comparing said first low resolution sequence representation and said second low resolution sequence representation to determine the presence of said target nucleic acid in said target sample.

33. The method of claim 32, wherein said sequence of at least two discrete nucleotides in said first low resolution sequence is the same as said sequence of at least two discrete nucleotides in said second low resolution sequence.

34. The method of claim 32, wherein a first plurality of low resolution sequence representations for a plurality of nucleic acids in said target sample are provided and a second plurality of low resolution sequence representations for a plurality of second nucleic acids in said reference sample are provided.

35. The method of claim 34, wherein said first low resolution sequence representation for said target nucleic acid and said second low resolution sequence representation for said second target nucleic acid are distinguished from low resolution sequence representations in said first plurality and in the second plurality.

36. The method of claim 35, further comprising quantifying the amount of the target nucleic acid in said target sample relative to the amount of the target nucleic acid in said reference sample.

37. The method of claim 32, wherein said first and second low resolution sequence representations have a known correlation with said actual sequence of said target nucleic acid at single nucleotide resolution.

38. The method of claim 32, wherein said first low resolution sequence representation and said second low resolution sequence representation are the same.

39. The method of claim 32, wherein said target nucleic acid has been bisulfite converted to replace cytosines with uracils.

40. The method of claim 39, wherein step (c) further comprises comparing said first low resolution sequence representation and said second low resolution sequence representation to determine the presence of said target nucleic acid in said target sample and to identify the location of a methylated cytosine in said target nucleic acid.

41. A method for determining the presence of a target nucleic acid in a sample, said method comprising the steps of:

(a) providing a barcode sequence from a target nucleic acid, wherein said target nucleic acid is obtained from said sample; and

(b) comparing said barcode sequence with a reference sequence, wherein the target nucleic acid is present in said sample if said reference sequence comprises a region corresponding to each determined region of the bar code sequence.

42. The method of claim 41 further comprising comparing the order of said determined regions of the bar code sequence with the order of corresponding regions in said reference sequence.

43. The method of claim 41 further comprising comparing the average distance between said determined regions of the bar code sequence with the average distance between corresponding regions in said reference sequence.

44. The method of claim 41, wherein said barcode sequence comprises a low resolution nucleic acid sequence representation.

45. The method of claim 44, wherein said low resolution nucleic acid sequence representation comprises an ordered series of determined regions.

46. The method of claim 45, wherein said low resolution nucleic acid sequence representation further comprises dark regions, wherein said dark regions are indicative of degenerate sequence composition, and wherein said dark regions intervene between said determined regions.

47. The method of claim 41, wherein said sample is a metagenomic sample.

48. The method of claim 41, wherein said reference sequence comprises a nucleic acid sequence.

49. The method of claim 41, wherein said reference sequence is present in a database of reference sequences.

50. The method of claim 49, wherein said reference sequences in said database are indexed by association with one or more groups of organisms.