Chase Ligation Sequencing

• US 20090191553A1
• Filed: 10/01/2008
• Published: 07/30/2009
• Est. Priority Date: 10/01/2007
• Status: Abandoned Application

• Current Assignee: Applied Biosystems LLC (Thermo Fisher Scientific Incorporated)
• Original Assignee: Applied Biosystems, Inc. (Thermo Fisher Scientific Incorporated)
• Inventors: Hendrickson, Cynthia
• Application Number:
  US12/243,925
• Publication Number:
  US 20090191553A1
• Time in Patent Office: Days
• US Class Current: 435/6.1
• CPC Class Codes: C12Q 1/6874 involving nucleic acid arrays, e.g. sequencing by hybridisation
• CPC Class Codes: C12Q 2525/161 incorporating target specific and non-target specific sites
• CPC Class Codes: C12Q 2525/185 incorporating bases where the precise position of the bases in the nucleic acid string is important
• CPC Class Codes: C12Q 2533/107 Probe or oligonucleotide ligation
• CPC Class Codes: C12Q 2537/155 Cyclic reactions

Abstract

In various embodiments, the present teachings provide sequencing methods which facilitate enhancing the efficiency of ligation and/or increasing sequencing reads. Various embodiments of the methods enable sequencing through template regions for which complementary labeled extension probes are unavailable or insufficient. In various embodiments, one or more rounds of ligation with unlabeled extension probes can be used in addition to a round of ligation with labeled extension probe. In various embodiments, for example, such methods can facilitate extension on template polynucleotides that do not bind labeled extension probe in the first round of ligation.

Claims

1. A method for determining a sequence of nucleotides in a template polynucleotide, the method comprising the steps of:

(a) providing a population of probe-template duplexes, each duplex comprising an initializing oligonucleotide probe hybridized to a template polynucleotide, the initializing oligonucleotide probe having an extendable terminus;

(b) ligating a labeled oligonucleotide extension probe to at least a fraction of the extendable termini of the population of probe-template duplexes to form labeled extended probe-template duplexes;

(c) ligating an unlabeled oligonucleotide extension probe to at least a fraction of the extendable termini of the population of probe-template duplexes that do not have a labeled oligonucleotide extension probe ligated thereto to form unlabeled extended probe-template duplexes;

(d) identifying after step (b) and before step (e) at least one nucleotide in the template polynucleotide;

(e) generating an extendable terminus on the oligonucleotide extension probe portion of at least a fraction of the labeled extended probe-template duplexes and the unlabeled extended probe-template duplexes; and

(f) repeating steps (b), (c), (d), and (e) until a sequence of nucleotides in the template polynucleotide is determined.

2. The method of claim 1, wherein the population of template nucleotides is attached to a microparticle.

3. The method of claim 2, wherein the microparticle is attached to a substrate.

4. The method of claim 3, wherein the microparticle is attached to the substrate by a linkage comprising biotin and a biotin-binding protein.

5. The method of claim 3, wherein the microparticle is not immobilized in a semi-solid support.

6. The method of claim 1, wherein the labeled oligonucleotide extension probes, the unlabeled oligonucleotide extension probes, or both comprise a scissile linkage.

7. The method of claim 6, wherein the scissile linkage is a phosphorothiolate linkage.

8. The method of claim 1, wherein the labeled oligonucleotide extension probes, the unlabeled oligonucleotide extension probes, or both have a non-extendable moiety at one terminus.

9. The method of claim 1, wherein the step of generating an extendable terminus on the oligonucleotide extension probe portion comprises the step of cleaving a phosphorothiolate linkage in the oligonucleotide extension probe with a cleavage agent comprising one or more Ag, Hg, Cu, Mn, Zn and Cd containing compounds.

10. The method of claim 9, wherein the cleavage agent comprises AgNO₃.

11. The method of claim 1, wherein the step of generating an extendable terminus on the oligonucleotide extension probe portion comprises generating an extendable terminus that is different from the extendable terminus to which the last oligonucleotide extension probe was ligated.

12. The method of claim 1, comprising a step of contacting the template polynucleotide with a blocking oligonucleotide prior to step (a).

13. The method of claim 1, wherein the step of identifying comprises detecting a label attached to the labeled oligonucleotide extension probe.

14. The method of claim 1, wherein the identified nucleotide is substantially complementary to the labeled oligonucleotide extension probe.

15. The method of claim 1, wherein the identified nucleotide is located within 1 residue of the labeled oligonucleotide extension probe.

16. The method of claim 1, wherein the step of identifying comprises detecting a detectable moiety attached by a cleavable linker to the labeled oligonucleotide extension probe.

17. The method of claim 16, wherein the cleavable linker comprises a disulfide bond.

18. The method of claim 1, wherein one or more steps comprise using bovine serum albumin to facilitate reducing the amount of ligase used.

19. The method of claim 1, wherein step (c) is repeated one or more times prior to step (e).

20. The method of claim 1, further comprising the steps of:

(g) removing the ligated oligonucleotide extension probes and the initializing oligonucleotide probes from the template polynucleotide;

(h) generating a population of probe-template duplexes, each duplex comprising a second initializing oligonucleotide probe hybridized to a template polynucleotide, the second initializing oligonucleotide probe being different from the previous initializing oligonucleotide probe and having an extendable terminus; and

(i) repeating steps (b), (c), (d), (e) and (f).

21. The method of claim 20, wherein steps (g), (h) and (i) are repeated one or more times, each time using an initializing oligonucleotide probe bound to a different sequence of the template polynucleotide.

22. A method for determining information about a sequence of nucleotides in a template polynucleotide using a first collection of at least 2 distinguishably labeled extension probe families, the method comprising the steps of.

(a) providing a population of probe-template duplexes, each duplex comprising an initializing oligonucleotide probe hybridized to a template polynucleotide, the initializing oligonucleotide probe having an extendable terminus;

(b) contacting the population of probe-template duplexes with a mixture of labeled oligonucleotide extension probes from at least 2 distinguishably labeled oligonucleotide extension probe families to ligate a labeled oligonucleotide extension probe to at least a fraction of the extendable termini of the population of probe-template duplexes to form labeled extended probe-template duplexes;

(c) ligating an unlabeled oligonucleotide extension probe to at least a fraction of the extendable termini of the population of probe-template duplexes that do not have a labeled oligonucleotide extension probe or a second labeled oligonucleotide extension probe ligated thereto to form unlabeled extended probe-template duplexes;

(d) detecting after step (b) and before step (e) the labels of at least a portion of the labeled extended probe-template duplexes;

(e) generating an extendable terminus on the oligonucleotide extension probe portion of at least a fraction of the labeled extended probe-template duplexes and the unlabeled extended probe-template duplexes;

(f) eliminating after step (d) and before step (g) one or more possibilities for the sequence of nucleotides in the template polynucleotide portion of the labeled extended probe-template duplexes based at least on the labels detected in step (d) to produce a list of potential sequences; and

(g) repeating steps (b), (c), (d), (e), and (f) until a sequence of nucleotides in the template polynucleotide is determined.

23. The method of claim 22, wherein the population of template nucleotides is attached to a microparticle.

24. The method of claim 23, wherein the microparticle is attached to a substrate.

25. The method of claim 24, wherein the microparticle is attached to the substrate by a linkage comprising biotin and a biotin-binding protein.

26. The method of claim 24, wherein the microparticle is not immobilized in a semi-solid support.

27. The method of claim 22, wherein the labeled extension probes comprise a constrained portion in which nucleotides are not independently selected, and wherein the labeled extension probes having constrained portions that differ in sequence are assigned to probe families according to an encoding.

28. The method of claim 27, wherein the determination of a sequence of the template polynucleotide comprises assigning detected labels to one of a first labeled oligonucleotide extension probe family, a second labeled oligonucleotide extension probe family, a third labeled oligonucleotide extension probe family, and a fourth labeled oligonucleotide extension probe family according to one or more of the 24 encodings set forth in Table 1.

29. The method of claim 22, wherein the labeled oligonucleotide extension probes in each probe family have the structure 5′

-(XY)(N)_kN_B*-3′

or 3′

-(XY)(N)_kN_B*-5′

, wherein N represents any nucleoside, N_B represents a moiety that is not extendable by ligase, * represents a detectable moiety, XY is a constrained portion of the probe in which X and Y represent nucleosides that are identical or different but are not independently selected, X and Y are at least 2-fold degenerate, at least one internucleoside linkage is a scissile linkage, and k is between 1 and 100, inclusive.

30. The method of claim 22, wherein the label associated with one or more labeled oligonucleotide extension probes comprises a combination of detectable moieties.

31. The method of claim 22, wherein the labeled oligonucleotide extension probes, the unlabeled oligonucleotide extension probes, or both comprise a scissile linkage.

32. The method of claim 31, wherein the scissile linkage is a phosphorothiolate linkage.

33. The method of claim 22, wherein the labeled oligonucleotide extension probes, the unlabeled oligonucleotide extension probes, or both have a non-extendable moiety at one terminus.

34. The method of claim 22, wherein the step of generating an extendable terminus on the oligonucleotide extension probe portion comprises the step of cleaving a phosphorothiolate linkage in the oligonucleotide extension probe with a cleavage agent comprising one or more Ag, Hg, Cu, Mn, Zn and Cd containing compounds.

35. The method of claim 22, wherein the step of generating an extendable terminus on the oligonucleotide extension probe portion comprises generating an extendable terminus that is different from the extendable terminus to which the last oligonucleotide extension probe was ligated.

36. The method of claim 22, comprising a step of contacting the template polynucleotide with a blocking oligonucleotide prior to step (a).

37. The method of claim 22, wherein the detecting step (d) comprises acquiring on average 2 bits of information substantially simultaneously from each of at least 2 nucleotides in the template polynucleotide without acquiring two bits of information from any individual nucleotide.

38. The method of claim 22, wherein the detecting step (d) comprises acquiring less than 2 bits of information simultaneously from each of at least 2 nucleotides in the template.

39. The method of claim 22, wherein the determination of a sequence of the template polynucleotide comprises:

(i) generating at least two candidate sequences from the list of potential sequences produced in two or more performances of step (f); and

(ii) selecting one of the at least two candidate sequences as the sequence of nucleotides in the template.

40. The method of claim 39, wherein the selecting step comprises:

(i) obtaining a second list of potential sequences for the template polynucleotide using a second collection of distinguishably labeled encoded probe families, wherein the probe families in the second collection of probe families are encoded differently to the probe families in the first collection of probe families;

(ii) generating at least one comparison sequence from the second ordered list of probe family names;

(iii) comparing a portion of at least one of the candidate sequences with a portion of at least one of the comparison sequences; and

(iv) selecting as the sequence of nucleotides in the template polynucleotide a candidate sequence that exhibits a predetermined level of identity, is most nearly identical to a comparison sequence, or both over the portion compared in step (iii).

41. The method of claim 40, wherein the portion compared is a single dinucleotide.

42. The method of claim 22, wherein the step of detecting comprises detecting a detectable moiety attached by a cleavable linker to the labeled oligonucleotide extension probe.

43. The method of claim 22, wherein one or more steps comprise using bovine serum albumin to facilitate reducing the amount of ligase used.

44. The method of claim 22, wherein step (c) is repeated one or more times prior to step (d).