Allele assignment and probe selection in multiplexed assays of polymorphic targets
First Claim
1. A method for reducing erroneous allele assignments where assignment is made based on the results of a hybridization assay between oligonucleotide probes and oligonucleotide targets, and where several polymorphic loci of interest are present on each allele, comprising:
- (i) selecting a set of primers for generating targets derived from genomic regions which include the polymorphic loci;
(ii) selecting a set of probes capable of hybridizing to subsequences in the targets, where the subsequences include nucleotides which are either complementary to or the same as a particular polymorphic locus;
(iii) determining whether the selected probes will—
when placed under suitable hybridization conditions with targets and where hybridization between probes including a particular sequence, and a particular subsequence, is detectable as a reaction (and where the detectable reactions of the probes and the subsequences forms a reaction pattern)—
generate an ambiguous reaction pattern consistent with more than one combination of two or more known alleles, and (a) if there is no ambiguity, selecting the probe set for analysis of samples from subjects; and
(b) if there is ambiguity, selecting a different set of probes in step (ii) and repeating step (iii) to attempt to eliminate the ambiguity;
but if the ambiguity cannot be eliminated, repeating step (i) to (iii) using a different set of primers.
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Abstract
A method to select a set of probes for multiplexed hybridization analysis of genes with multiple polymorphic regions, which minimizes ambiguities (where the assay results can correspond with more than one allele combination) by one or more of several methods, including: eliminating probes which generate ambiguities; setting a threshold such that only probe-target interactions above the threshold are considered as positive; selectively adding probes until ambiguities are eliminated.
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Citations
28 Claims
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1. A method for reducing erroneous allele assignments where assignment is made based on the results of a hybridization assay between oligonucleotide probes and oligonucleotide targets, and where several polymorphic loci of interest are present on each allele, comprising:
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(i) selecting a set of primers for generating targets derived from genomic regions which include the polymorphic loci;
(ii) selecting a set of probes capable of hybridizing to subsequences in the targets, where the subsequences include nucleotides which are either complementary to or the same as a particular polymorphic locus;
(iii) determining whether the selected probes will—
when placed under suitable hybridization conditions with targets and where hybridization between probes including a particular sequence, and a particular subsequence, is detectable as a reaction (and where the detectable reactions of the probes and the subsequences forms a reaction pattern)—
generate an ambiguous reaction pattern consistent with more than one combination of two or more known alleles, and (a) if there is no ambiguity, selecting the probe set for analysis of samples from subjects; and
(b) if there is ambiguity, selecting a different set of probes in step (ii) and repeating step(iii) to attempt to eliminate the ambiguity;
but if the ambiguity cannot be eliminated, repeating step (i) to (iii) using a different set of primers. - View Dependent Claims (2, 3, 4, 5, 6, 7, 8, 9, 10, 13, 24, 25, 26, 27, 28)
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11. A method for reducing erroneous allele assignments where assignment is made based on the results of a hybridization assay between oligonucleotide probes and oligonucleotide targets (where the targets are derived from and/or include subsequences complementary to or the same as subsequences in selected alleles, and where the subsequences in the selected alleles include several polymorphic loci) by making allele assignments where mismatches between probes and targets as observed in the hybridization assay, as compared with mismatches predicted between probes and targets, occur at less than a predetermined frequency, comprising:
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(i) selecting a set of probes capable of hybridizing to the targets;
(ii) assaying by placing the probes in contact with the targets under hybridizing conditions where hybridization between probes including a particular sequence, and a particular subsequence of the targets, is detectable as a reaction signal of a particular intensity, wherein the intensity is proportional to said hybridizations, and where the detectable signals from reactions of the probes and the target subsequences forms a reaction pattern;
(iii) determining a reference threshold, T, for probes including a particular sequence using the following algorithm;
Ti=Rmin+(Rmax−
Rmin)* i/X
Si=(Σ
((Rk−
Ti)* σ
k)/Σ
|((Rk−
Ti)|
T=Max (Si)Where;
k ranges from 1 to N, and N is the number of probes in the set of probes;
σ
k=1, when reaction is positive;
σ
k=−
1, when reaction is negative;
i ranges from 1 to X;
Rk is the ratio of the probe'"'"'s intensity over a known positive control probe intensity;
Rmax and Rmin are the respective maximum and minimum values for this ratio; and
Ti is a calculated threshold for a probe-target interaction;
(iv) including in the reaction pattern only the signals having intensity greater than or equal to the threshold;
(v) determining the predicted reaction pattern produced by predicting reaction of the probe set with predicted targets which are predicted to be generated by derivation of known allele combinations; and
(vi) comparing the reaction pattern generated by the assay with the predicted reaction pattern, and assigning alleles only if the mismatches between the two patterns occurs at a frequency less than or equal to a specified tolerance level. - View Dependent Claims (12, 14, 15, 16, 17, 18, 19)
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20. A method for reducing erroneous allele assignments where assignment is made based on the results of a hybridization assay between oligonucleotide probes and oligonucleotide targets, and where several polymorphic loci of interest are present on each allele, comprising:
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(i) selecting a set of primers for generating derived targets from genomic regions which include the polymorphic loci;
(ii) selecting an initial set of probes capable of hybridizing to subsequences in the targets, where the subsequences include nucleotides which are either complementary to or the same as particular polymorphic loci;
(iii) selecting a core probe subset from the initial probe set;
(iv) determining whether the core probe set will—
when placed under suitable hybridization conditions with targets and where hybridization between probes including a particular sequence, and a particular subsequence, is detectable as a reaction (and where the detectable reactions of the probes and the subsequences forms a reaction pattern)—
generate an ambiguous reaction pattern consistent with more than one combination of two or more known alleles, and (a) if there is no ambiguity, or if the ambiguity is acceptable, selecting the core probe set for analysis of samples from subjects;
but (b) if the ambiguity is unacceptable, adding selected probes from the initial probe set to the core probe set and repeating step (iv) following additions to attempt to bring the ambiguity to an acceptable level. - View Dependent Claims (21, 22, 23)
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Specification