Enhanced Sequencing by Hybridization Using Pools of Probes
First Claim
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.
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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.
2 Citations
47 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.
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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, - View Dependent Claims (7, 8, 9, 10)
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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, - View Dependent Claims (12, 13)
- 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
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14. A method of detecting one or more sequences in a nucleic acid target comprising:
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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 - View Dependent Claims (15, 16, 17, 18, 19, 20)
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21. A method of detecting one or more sequences in a plurality of nucleic acid targets comprising:
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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. - View Dependent Claims (22, 23, 24)
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25. A method for determining a sequence of a nucleic acid target comprising:
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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. - View Dependent Claims (26, 27, 28, 29)
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30. A method for determining a sequence of a nucleic acid target comprising:
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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 - View Dependent Claims (31)
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32. A method for determining a sequence of a nucleic acid target comprising:
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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. - View Dependent Claims (33, 34)
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35. A method for determining a sequence of a nucleic acid target comprising:
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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
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- 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.
- 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.
Specification