Enhanced sequencing by hybridization using pools of probes
First Claim
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1. A method of identifying one or more sequences of a target nucleic acid comprising:
- a. contacting a target nucleic acid with a first set of pools of probes, wherein at least one pool in the set comprises a mixture of two or more probes having different sequences in information regions of the probes, under conditions which produce, on average, more probe;
target hybridization with probes which are perfectly complementary to the target nucleic acid in the information region of the probes than with probes which are mismatched in the information regions;
b. detecting a first subset of pools for which a level of hybridization indicates that there is at least one perfectly complementary probe within each pool; and
c. identifying one or more sequences of the target nucleic acid from the first subset of pools detected in step (b) by compiling overlapping sequences of the information regions of the probes in the subset of detected pools, wherein one or more pooling false positive probes are eliminated as a result of compilation of overlapping sequences.
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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.
245 Citations
35 Claims
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1. A method of identifying one or more sequences of a target nucleic acid comprising:
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a. contacting a target nucleic acid with a first set of pools of probes, wherein at least one pool in the set comprises a mixture of two or more probes having different sequences in information regions of the probes, under conditions which produce, on average, more probe;
target hybridization with probes which are perfectly complementary to the target nucleic acid in the information region of the probes than with probes which are mismatched in the information regions;
b. detecting a first subset of pools for which a level of hybridization indicates that there is at least one perfectly complementary probe within each pool; and
c. identifying one or more sequences of the target nucleic acid from the first subset of pools detected in step (b) by compiling overlapping sequences of the information regions of the probes in the subset of detected pools, wherein one or more pooling false positive probes are eliminated as a result of compilation of overlapping sequences. - View Dependent Claims (2, 3, 4, 12, 13, 14, 15, 16, 17, 18, 19)
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5. A method of identifying one or more sequences of a target nucleic acid comprising:
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a. contacting a target nucleic acid with a first set of pools of probes, wherein at least one pool in the set comprises a mixture of two or more probes having different sequences in information regions of the probes, under conditions which produce, on average, more probe;
target hybridization with probes which are perfectly complementary to the target nucleic acid in the information region of the probes than with probes which are mismatched in the information regions;
b. assigning a hybridization score to each probe wherein each probe within a pool is assigned the same hybridization score; and
c. identifying one or more sequences of the target nucleic acid by analysis of hybridization scores of overlapping probes, wherein one or more probes with false high scores arising from pooling of probes are eliminated by analysis of hybridization scores of overlapping probes. - View Dependent Claims (6, 7, 8, 9, 10, 11)
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20. A method of identifying one or more sequences of a target nucleic acid comprising:
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a. contacting a target nucleic acid with a first set of pools of immobilized probes and a first set of pools of labeled probes, wherein at least one pool in either the first set of pools of immobilized probes, or in the first set of pools of labeled probes, or in both, comprises a mixture of two or more probes having different sequences in the information regions of the probes, under conditions which produce, on average, more probe;
target hybridization for probes which are perfectly complementary to the target nucleic acid in the information region than with probes which are mismatched in the information region;
b. covalently joining adjacently hybridized immobilized probes and labeled probes to provide a first set of covalently joined probes;
c. detecting a first subset of pools of covalently joined probes for which a level of hybridization indicates that there is at least one perfectly complementary covalently joined probe within each pool; and
d. identifying one or more sequences of the target nucleic acid from the first subset of covalently joined pools of probes detected in step (c) by compiling overlapping sequences of the information regions of covalently joined probes in the subset of detected pools, wherein one or more covalently joined pooling false positive probes are eliminated as a result of compilation of overlapping sequences. - View Dependent Claims (21, 25, 26, 27, 28, 29, 30, 31, 34, 35)
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22. A method of identifying one or more sequences of a target nucleic acid comprising:
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a. contacting a target nucleic acid with a first set of pools of immobilized probes and a first set of pools of labeled probes, wherein at least one pool in either the first set of pools of immobilized probes, or in the first set of pools of labeled probes, or in both, comprises a mixture of two or more probes having different sequences in the information regions of the probes, under conditions which produce, on average, more probe;
target hybridization for probes which are perfectly complementary to the target nucleic acid in the information region than with probes which are mismatched in the information region;
b. covalently joining adjacently hybridized immobilized probes and labeled probes to provide a first set of covalently joined probes;
c. assigning a hybridization score to each covalently joined probe in the first set wherein each probe within a pool of covalently joined probes is assigned the same hybridization score; and
e. identifying one or more sequences of the target nucleic acid from overlapping covalently joined probes by analysis of hybridization scores of overlapping covalently joined probes wherein one or more covalently joined probes with false high scores arising from pooling of probes are eliminated by analysis of hybridization scores of overlapping probes. - View Dependent Claims (23, 24, 32, 33)
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Specification