Methods of validating SNPs and compiling libraries of assays
OwnerFrancisco M. De La Vega, Janet S. Ziegle +4inventors
InventorsFrancisco M. De La Vega, Janet S. Ziegle, Hadar I. Avi-Itzhak, Charles R. Scafe, Eugene G. Spier, Yu N. Wang
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Abstract
Libraries of assays and methods of compiling the libraries are provided. The assays can identify Single Nucleotide Polymorphisms (SNPs). Methods of validating SNPs are provided. Methods of constructing linkage disequilibrium maps using sets or subsets of SNPs are also provided.
Claims34
A method of compiling a library of polynucleotide data sets that correspond to polynucleotides that each can function as (A) a primer for producing a nucleic acid sequence that is complementary to at least one target nucleic acid sequence including a target Single Nucleotide Polymorphism (SNP), (B) a probe for rendering detectable the at least one target nucleic acid sequence including a target SNP, or (C) both (A) and (B), the method comprising the steps of:
- selecting for the library polynucleotide data sets that each correspond to a respective polynucleotide that contains a sequence that is complementary to a respective first allele included in each of the at least one target nucleic acid sequences, if, under a set of reaction conditions:
- (1) the respective polynucleotide has a background signal value less than or equal to a first defined value, where the background signal value is a first normalized ratio of a fluorescence intensity of the respective polynucleotide reacted with first assay reactants in the absence of the target nucleic acid sequence, and under first conditions of fluorescence excitation, to a dye fluorescence intensity of a passive-reference dye under the first conditions;
- (2) the respective polynucleotide has a signal generation value of greater than or equal to a second defined value, wherein the signal generation value is the difference between (i) a second normalized ratio of the fluorescence intensity of the respective polynucleotide reacted with the first assay reactants in the presence of the target nucleic acid sequence, to the dye fluorescence intensity and (ii) the background signal value;
- (3) the respective polynucleotide has a specificity value of less than or equal to a third defined value, wherein the specificity value is the difference between (i) a third normalized ratio of the fluorescence intensity of the respective polynucleotide reacted with second assay reactants that contain a second allele included in the at least one target nucleic acid sequence to the dye fluorescence intensity, wherein the second allele differs from the first allele, and (ii) the background signal value;
- (4) at least one individual from a population of individuals has a genotype identifiable under the first conditions, that results from reacting the respective polynucleotide with the first assay reactants and in the presence of the target nucleic acid sequence, wherein the population includes at least one individual that has the identifiable genotype and at least one individual that does not have the identifiable genotype;
- and (5) at least one individual from the population has an identifiable minor allele of the identifiable genotype, under the first conditions that results from reacting the respective polynucleotide with the first assay reactants in the presence of the target nucleic acid sequence, wherein the population includes at least one individual that has the identifiable minor allele, and at least one individual that does not have the identifiable minor allele.
A method of compiling a library of polynucleotide data sets that correspond to polynucleotides that each can function as (A) a primer for producing a nucleic acid sequence that is complementary to at least one target nucleic acid sequence including a target SNP, (B) a probe for rendering detectable the at least one target nucleic acid sequence including a target SNP, or (C) both (A) and (B), the method comprising the steps of:
- (1) determining a background signal value by calculating a first normalized ratio of a fluorescence intensity of a respective polynucleotide that contains a sequence that is complementary to a first allele included in the at least one target nucleic acid sequence, reacted with first assay reactants in the absence of the target nucleic acid sequence, and under first conditions of fluorescence excitation, to a dye fluorescence intensity of a passive-reference dye under the first conditions;
- (2) comparing a difference between (i) a second normalized ratio of the fluorescence intensity of the respective polynucleotide reacted with the first assay reactants in the presence of the target nucleic acid sequence, to the dye fluorescence intensity, and (ii) the background signal value;
- (3) comparing a difference between (i) a third normalized ratio of the fluorescence intensity of the respective polynucleotide reacted with second assay reactants that contain a second allele included in the at least one target nucleic acid sequence to the dye fluorescence intensity, wherein the second allele differs from the first allele, and (ii) the background signal value;
- (4) determining whether at least one individual from a population of individuals has a genotype identifiable under the first conditions that results from reacting the respective polynucleotide with the first assay reactants and in the presence of the target nucleic acid sequence, wherein the population includes at least one individual that has the identifiable genotype and at least one individual that does not have the identifiable genotype;
- and
- (5) determining whether at least one individual from the population has an identifiable minor allele of the identifiable genotype, under the first conditions that results from reacting the respective polynucleotide with the first assay reactants in the presence of the target nucleic acid sequence.
A method of confirming the existence of a SNP, the method comprising the steps of:
- identifying a location corresponding to a possible SNP in a polynucleotide in a first collection of data sets containing information on genomic deoxyribonucleic acid (DNA) samples in the form of data sets corresponding to polynucleotides;
- and
- confirming the existence of the SNP if at least one of the following conditions is met:
- (1) a second collection of data sets containing information on genomic deoxyribonucleic acid (DNA) samples contains information that identifies the location as containing the possible SNP;
- (2) at least two data sets from the first collection of data sets contain information corresponding to a minor allele of the possible SNP at the location, wherein the at least two data sets represent genomic deoxyribonucleic acid (DNA) samples obtained from two independent sources;
- and
- (3) a data set that corresponds to a consensus sequence of genomic deoxyribonucleic acid (DNA) samples in a third collection of data sets contains the minor allele of the possible SNP, wherein a source of the consensus sequence of genomic deoxyribonucleic acid (DNA) samples and sources of the genomic deoxyribonucleic acid (DNA) samples from the first collection of data sets are independent.
A library that contains data corresponding to respective oligonucleotides that can function as assays to detect Single Nucleotide Polymorphisms (SNPs), the library comprising a number of data sets corresponding to not more than a sufficient number of oligonucleotides necessary to provide a collection of assays that provides a maximum statistical loss of haplotype diversity, across a human genome, of less than ten (10) percent.
Dependencies are inferred from the claim text.