Enhanced Sequencing by Hybridization Using Pools of Probes

• US 20090280481A1
• Filed: 10/30/2007
• Published: 11/12/2009
• Est. Priority Date: 01/06/1999
• Status: Active Grant

• Current Assignee: Complete Genomics Incorporated (BGI Genomics Co., Ltd.)
• Original Assignee: Callida Genomics, Inc. (SBH Genomics, Inc.)
• Inventors: Drmanac, Radoje, Kita, David, Cooke, Cory, Drmanac, Snezana, Xu, Chongjun
• Granted Patent: US 8,034,566 B2
• Time in Patent Office: Days
• US Class Current: 435/6
• CPC Class Codes: C12Q 1/6874 involving nucleic acid arrays, e.g. sequencing by hybridisation
• CPC Class Codes: C12Q 2537/165 Mathematical modelling, e.g. logarithm, ratio

Abstract

The invention provides methods for sequencing by hybridization (SBH) using pools of probes that allow greater efficiency in conducting SBH by reducing the number of separate measurements of hybridization signals required to identify each particular nucleotide in a target nucleic acid sequence. The invention also provides pools and sets of pools of probes, as well as methods of generating pools of probes.

Claims

1. A nucleic acid assay comprising the step of testing a nucleic acid target with a set of probes of know informative sequence arranged in a plurality of probe pools, wherein probes with minimal allowed relatedness in the informative sequence are predominately included in the same pool.

2. The method of claim 1 wherein said set of probes comprises all or substantially all probes of a given informative length.

3. The method of claim 1 wherein at least some of said probe pools are labeled with different label and used as a pool mix.

4. The method of claim 1 further comprising finding two or more oligonucleotide sequences in two or more pools with significant score value or in two or more pools determined to be positive that match a sequence in the target

5. The method of claim 1, 2, 3 or 4 wherein pooled probes are ligated to other individual or pooled probes when two continuously hybridize to the target

6. A method of analyzing a target nucleic acid sequence comprising

a) providing a complete or substantially complete set of individually prepared oligonucleotides of a given number of informative bases, wherein at least two probes that differ at more than one position and that are either not labeled or labeled with the same label are pooled wherein substantially each probe is present only in one pool, thus reducing the number of measurements in testing the said set of oligonucleotides;

and

b) hybridize said pools to a target nucleic acid attached to a support or in solution thus analyzing some target bases multiple times with a number of oligonucleotide close or equal to said number of informative bases,

7. The method of claim 6 wherein multiple analyses per base reduces experimental errors.

8. The method of claim 6 wherein oligonucleotides with substantially maximal allowed sequence differences for a given set of oligonucleotides and given number of pools are pooled together.

9. The method of claim 6 wherein a pool has less oligonucleotides than a pool of the same consensus sequence prepared by single synthesis using two or more mixed nucleotides at positions that differ at least between two oligonucleotides.

10. The method of claim 6, 7, 8 or 9 further comprising ligation of pooled oligonucleotides to other oligonucleotides hybridized to said target.

11. A method of analyzing a target nucleic acid sequence comprising the steps of

a) providing a complete or substantially complete set of oligonucleotides of a given number of informative bases;

wherein at least two probes that differ at more then one positions and that are either not labeled or labeled with the same label are synthesized together as a pool, thus reducing the number of measurements in testing the said set of oligonucleotides and

c) hybridizing said pools to a target nucleic acid thus analyzing some target bases multiple times with a number of oligonucleotide close or equal to said number of informative bases,

12. The method of claim 11 wherein number of overlapping oligonucleotides is minimized per pool.

13. The method of claim 11 or 12 further comprising the step of ligating pooled oligonucleotides to other oligonucleotides hybridized to said target.

14. A method of detecting one or more sequences in a nucleic acid target comprising:

performing ligation of two sets of oligonucleotides of known informative sequence using a target nucleic acid as template, wherein at least one of said oligonucleotide sets is in the form of a predefined number of pools, at least some pools containing two or more oligonucleotides having different informative sequences and the same label, wherein none, some or all of said pools are combined if oligonucleotides in different pools are labeled with different labels, and

comparing ligation results of al least some of said pools

15. The method of claim 14 wherein oligonucleotides with minimal relatedness in the informative sequence are predominately included in the same pool.

16. The method of claim 14 wherein at least one set has all or substantially all oligonucleotides of given informative length.

17. The method of claim 14 wherein substantially each probe are present only in one pool.

18. The method of claim 14 wherein said targets or one set of probes are attached to one or more solid supports such as beads or other particles or glass slides.

19. The method of claim 14 wherein arrays of samples are used multiple times for testing different probes or probe pairs.

20. The method of claim 14, 17, or 18 wherein the mutated base is read at least with two probes from at least two pools.

21. A method of detecting one or more sequences in a plurality of nucleic acid targets comprising:

performing ligation of a set of oligonucleotide pools to oligonucleotide molecules present in support bound hybridization complexes of said oligonucleotide molecules and said nucleic acid targets, wherein each oligonucleotide pool contains two or more oligonucleotides not labeled or labeled with the same label and having different known informative sequence, and wherein none, some or all of said oligonucleotide pools are combined if oligonucleotides in different pools are labeled with different labels, and

comparing ligation results of at least some of said pools.

22. The method of claim 21 wherein the set of oligonucleotide pools comprises all or almost all probe sequences of a given informative length.

23. The method of claim 22 where substantially each probes are present only in one pool.

24. The method of claim 21 wherein target nucleic acid is attached to a solid support.

25. A method for determining a sequence of a nucleic acid target comprising:

a) providing a set of measured values for a nucleic acid target wherein

i) one value represent a pool of different oligonucleotide sequences,

ii) higher values indicate higher chance that one or more oligonucleotide sequences from such pool is present in the said target, and

iii) majority of oligonucleotide sequences in majority of said pools is not present in said target;

and

b) determining a sequence of said target by finding two or more oligonucleotide sequences from two or more pools with significant value that match said sequence of said target.

26. The method of claim 25 wherein in part (b) further comprises eliminating some oligonucleotide sequences from a pool as experimental false positive.

27. The method of claim 26 wherein eliminated sequences do not match a reference sequence or match or overlap other oligonucleotide sequence.

28. The method of claim 25 wherein in part (ii) lower values indicate higher chance.

29. The method of claim 25 wherein substantially each oligonucleotide sequence is present in a single pool.

30. A method for determining a sequence of a nucleic acid target comprising:

a) providing a set of measured values for a nucleic acid target wherein

i) one value represent a pool of different oligonucleotide sequences,

ii) higher values indicate higher chance that one or more oligonucleotide sequences from such pool is present in the said target, and

iii) majority of oligonucleotide sequences in majority of said pools is not present in said target; and

b) determining a sequence of said target by comparing oligonucleotide sequences from different pools with significant values

31. The method of claim 30 wherein a consensus sequence of at least one pool of oligonucleotides has multiple or three or four letters from letters V, H, D, B and N.

32. A method for determining a sequence of a nucleic acid target comprising:

a) providing a set of measured values for a nucleic acid target wherein

i) one value represent a pool of different oligonucleotide sequences,

ii) higher values indicate higher chance that one or more oligonucleotide sequences from such pool is present in the said target, and

iii) majority of oligonucleotide sequences in majority of said pools is not present in said target; and

b) determining a sequence of said target by finding overlapping oligonucleotide sequences form different pools with significant values.

33. The method of claim 32 wherein in part (b) further comprises eliminating some oligonucleotide sequences from a pool as experimental false positive.

34. The method of claim 32 further comprising the step of obtaining said measured values comprises preparing a pool of oligonucleotides by mixing independently synthesized oligonucleotides of different informative sequence all unlabeled or all labeled with the same label such as a fluorescent dye wherein substantively each oligonucleotide is added in a single pool.

35. A method for determining a sequence of a nucleic acid target comprising:

a) providing a set pool of different oligonucleotide sequences expected to have

i) at least one oligonucleotide sequence present in analyzed target, and

ii) majority of oligonucleotide sequences in majority of said pools not present in said target; and

b) determining a sequence of said target by finding two or more oligonucleotide sequences from two or more said pools that match said sequence of said target

36. A complete or almost complete set of oligonucleotides of known sequence of given number of informative bases that are prepared individually in independent synthesis reactions and then mixed in a set of pools wherein oligonucleotides in a pool are i) not labeled or labeled with the same label and ii) differ at more than one informative position.

37. The set of claim 36 wherein substantively each oligonucleotide is added in a single pool.

38. The set of claim 36 wherein a pool have less oligonucleotides than a pool of the same consensus sequence prepared by single synthesis using two or more mixed nucleotides at positions that differ at least between two oligonucleotides.

39. The set of claim 36 wherein each prepared oligonucleotide has one or more degenerated or universal bases.

40. The set of claim 36 wherein pools labeled with different labels are mixed together.

41. A computer system comprising software that analyses data obtained using the set of pools of claim 36.

42. The system of claim 41 for use in eliminating experimental or pool false positives.

43. A system comprising one or more containers with said pools according to claim 36 and a legating agent separated from or mixed with pools.

44. A set of oligonucleotides of known informative sequence arranged in two or more pools wherein oligonucleotides with minimal relatedness or maximal difference in the informative sequences are predominately included in the same pool, and wherein none, some or all of said pools are combined when oligonucleotides in different pools are labeled with different labels.

45. The set of claim 44 wherein pools are prepared by mixing individually prepared oligonucleotides or smaller pools of oligonucleotides.

46. The set of claim 44 wherein said oligonucleotides have two informative bases.

47. The set of claim 44 wherein at least some of said probe pools if labeled with different label are combined in a mix.