EP1483405A2 - Procedes de validation de snp et de compilation de banques de dosages - Google Patents

Procedes de validation de snp et de compilation de banques de dosages

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Publication number
EP1483405A2
EP1483405A2 EP03707532A EP03707532A EP1483405A2 EP 1483405 A2 EP1483405 A2 EP 1483405A2 EP 03707532 A EP03707532 A EP 03707532A EP 03707532 A EP03707532 A EP 03707532A EP 1483405 A2 EP1483405 A2 EP 1483405A2
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EP
European Patent Office
Prior art keywords
snps
library
snp
nucleic acid
polynucleotide
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Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
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EP03707532A
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German (de)
English (en)
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EP1483405A4 (fr
Inventor
Francisco De La Vega
Yu N. Wang
Eugene G. Spier
Charles R. Scafe
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Applied Biosystems Inc
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Applera Corp
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Priority claimed from US10/335,690 external-priority patent/US20040063109A2/en
Priority claimed from US10/335,707 external-priority patent/US20030190652A1/en
Application filed by Applera Corp filed Critical Applera Corp
Publication of EP1483405A2 publication Critical patent/EP1483405A2/fr
Publication of EP1483405A4 publication Critical patent/EP1483405A4/fr
Withdrawn legal-status Critical Current

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6813—Hybridisation assays
    • C12Q1/6827—Hybridisation assays for detection of mutation or polymorphism

Definitions

  • Assays can include probes and/or primers that hybridize with a target nucleic acid sequence. These probes and primers can be useful for visualizing or amplifying the target nucleic acid sequence.
  • the target nucleic acid sequence can have a Single Nucleotide Polymorphism (SNP) contamed therein.
  • SNP Single Nucleotide Polymorphism
  • a relative indication of concentration of the target nucleic acid sequence can be obtained based on relative fluorescence of the probes.
  • SNPs Single nucleotide polymorphisms
  • SNPs are promising tools for mapping susceptibility mutations that contribute to complex diseases. Although most SNPs are neutral and do not affect phenotype, they can be used as surrogate markers for positional cloning of genetic loci, because of the allelic association, known as linkage disequilibrium (LD), that can be shared by groups of adjacent SNPs. LD is eroded by gene conversion and recombination, and the amount of LD depends on the age of the mutations and on the demographic history of the population. The extent of LD across a genomic region dictates the density of SNP markers necessary to ensure association between a marker and the causative allele sought.
  • LD linkage disequilibrium
  • methods are provided for SNP validation that take into consideration a number of findings and statistics. Studies have reported a discontinuous structure in the patterns of LD across a set of regions sampled from the human genome, where long stretches of strong LD are punctuated by recombination hot-spots. These LD "blocks" show little evidence of historical recombination. According to various embodiments, these results are deconvoluted to predict that a reduced set of contiguous chromosomal segments, or haplotypes, exist in specific populations.
  • haplotype diversity is made up on only 4 to 6 so-called common haplotypes.
  • LD block patterns change depending on the population sampled because of historical differences; for example, populations that have experienced bottlenecks (e.g., Caucasians) show longer LD blocks and less evidence of historical recombination events, than other populations.
  • the haplotype diversity in a given population is typically constant in a given region irrespective of the number of SNPs sampled; therefore typing an arbitrarily large number of SNPs within a LD block is unnecessary. Selecting the minimum subset of SNPs within LD blocks, or any other discrete genetic locus, that enable discrimination of the common haplotypes present in a block without loss of information can be used to validate SNPs and/or to compile a concise library of assays useful for genetic analysis.
  • a method of compiling a library of polynucleotide data sets can 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 can include the step 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 a number of parameters are met by each polynucleoti.de corresponding to the data sets included in the library.
  • the parameters can include: (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
  • a method of compiling a library of polynucleotide data sets can 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 can include the step of 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.
  • the method can include the step of 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.
  • the method can include the step of 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.
  • the method can include the step of 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.
  • the method can include the step of 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.
  • Various combinations of the herein described method steps and/or parameters can be used.
  • a method of confirming the existence of a SNP can include the step 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.
  • the method can include the step of confirming the existence of the SNP if at least one condition is met. A condition can be met if 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.
  • a condition can be met, for example, if 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.
  • a condition can be met if a data set that corresponds to a consensus sequence of genomic deoxyribonucleic acid (DNA) samples that contains the minor allele of the possible SNP in a third collection of data sets.
  • the source of the consensus sequence of genomic deoxyribonucleic acid (DNA) samples, and the sources of the genomic deoxyribonucleic acid (DNA) samples from the first collection of data sets, can be independent.
  • a library contains data corresponding to respective oligonucleotides that can function as assays to detect Single Nucleotide Polymorphisms (SNPs).
  • the library can have 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 a defined percentage of haplotype diversity across a human genome.
  • an algorithm to select the minimal subset of SNPs required for capturing the diversity of haplotype blocks or other genetic loci is provided.
  • the algorithm can be used to quickly select the minimum SNP subset with no loss of haplotype information.
  • the algorithm can be used in a more aggressive mode to further reduce the original SNP set, with minimal loss of information.
  • Figs, la-b are graphs of SNPs per LD block v. minimum information SNP subset for African- American and Caucasian populations, respectively;
  • Figs. 2a-2e are schematic diagrams of quenchable dyes that can be part of a mixture of reagents provided and/or used according to various embodiments;
  • Fig. 3 is a workflow diagram according to various embodiments.
  • Fig. 4 is a graph showing visualized assay results, according to various embodiments.
  • Fig. 5 is a flowchart showing an algorithm according to various embodiments
  • Fig. 6 is a flowchart showing an algorithm according to various embodiments
  • Fig. 7 is an illustration of SNPs selected by a method according to various embodiments.
  • Fig. 8 shows a table of SNPs and genes on a hypothetical chromosome
  • Fig. 9 is a histogram of gene lengths of the hypothetical chromosome of Fig. 8;
  • Fig. 10 is a histogram of the specified maximum distance between adjacent SNPs;
  • Fig. 11 is a histogram of actual maximum distance between adjacent SNPs
  • Fig. 12 is a histogram of total selected SNPs per gene.
  • nucleic acid analogs can be used in addition to or instead of nucleic acids.
  • nucleic acid analogs can include the family of peptide nucleic acids (PNA), wherein the sugar/phosphate backbone of DNA or RNA has been replaced with acyclic, achiral, and neutral polyamide linkages.
  • PNA peptide nucleic acids
  • a probe or primer can have a PNA polymer instead of a DNA polymer.
  • the 2-aminoethylglycine polyamide linkage with nucleobases attached to the linkage through an amide bond can be used as a PNA and shown to possess exceptional hybridization specificity and affinity.
  • An example of a PNA is as shown below in a partial structure with a carboxyl-terminal amide:
  • Nucleobase as used herein means any nitrogen-containing heterocyclic moiety capable of forming Watson-Crick hydrogen bonds in pairing with a complementary nucleobase or nucleobase analog, e.g. a purine, a 7-deazapurine, or a pyrimidine.
  • Typical nucleobases are the naturally occurring nucleobases such as, for example, adenine, guanine, cytosine, uracil, thymine, and analogs of the naturally occurring nucleobases, e.g.
  • Nucleoside refers to a compound consisting of a nucleobase linked to the C-1' carbon of a sugar, such as, for example, ribose, arabinose, xylose, and pyranose, in the natural ⁇ or the ⁇ anomeric configuration.
  • the sugar can be substituted or unsubstituted.
  • Substituted ribose sugars can include, but are not limited to, those riboses having one or more of the carbon atoms, for example, the 2'-carbon atom, substituted with one or more of the same or different Cl, F 5 -R, -OR, -NR 2 or halogen groups, where each R is independently H, Ct-C ⁇ alkyl or C 5 -C 1 aryl.
  • Ribose examples can include ribose, 2'- deoxyribose, 2',3'-dideoxyribose, 2'-haloribose, 2'-fluororibose, 2'-chlororibose, and 2'- alkylribose, e.g. 2'-O-methyl, 4'- ⁇ -anomeric nucleotides, l'- ⁇ -anomeric nucleotides, 2'-4'- and 3'-4'-linked and other "locked" or "LNA", bicyclic sugar modifications.
  • Exemplary LNA sugar analogs within a polynucleotide can include the following structures:
  • Sugars can have modifications at the 2'- or 3'-position such as methoxy, ethoxy, allyloxy, isopropoxy, butoxy, isobutoxy, methoxyethyl, alkoxy, phenoxy, azido, amino, alkylamino, fluoro, chloro and bromo.
  • Nucleosides and nucleotides can have the natural D configurational isomer (D-form) or the L configurational isomer (L-form).
  • the nucleobase is a purine, e.g. adenine or guanine
  • the ribose sugar is attached to the N - position of the nucleobase.
  • the nucleobase is a pyrimidine, e.g. cytosine, uracil, or thymine
  • the pentose sugar is attached to the N -position of the nucleobase.
  • Nucleotide refers to a phosphate ester of a nucleoside and can be in the form of a monomer unit or within a nucleic acid.
  • Nucleotide 5'-triphosphate refers to a nucleotide with a triphosphate ester group at the 5' position, and can be denoted as “NTP", or “dNTP” and “ddNTP” to particularly point out the structural features of the ribose sugar.
  • the triphosphate ester group can include sulfur substitutions for the various oxygens, e.g. ⁇ -thio-nucleotide 5'-triphosphates.
  • polynucleotide and “oligonucleotide” mean single-stranded and double-stranded polymers of, for example, nucleotide monomers, including 2'- deoxyribonucleotides (DNA) and ribonucleotides (RNA) linked by intemucleotide phosphodiester bond linkages, e.g. 3'-5' and 2'-5', inverted linkages, e.g. 3 '-3' and 5 '-5', branched structures, or intemucleotide analogs.
  • Polynucleotides can have associated counter ions, such as H , NH , trialkylammonium, Mg , Na and the like.
  • a polynucleotide can be composed entirely of deoxyribonucleotides, entirely of ribonucleotides, or chimeric mixtures thereof.
  • Polynucleotides can be comprised of intemucleotide, nucleobase and sugar analogs.
  • a polynucleotide or oligonucleotide can be a PNA polymer.
  • Polynucleotides can range in size from a few monomeric units, e.g. 5-40 when they are more commonly frequently referred to in the art as oligonucleotides, to several thousands of monomeric nucleotide units.
  • “Intemucleotide analog” as used herein means a phosphate ester analog or a non- phosphate analog of a polynucleotide.
  • Phosphate ester analogs can include: (i) C 1 -C all y lphosphonate, e.g. methylphosphonate; (ii) phosphoramidate; (iii) Ci-C 6 alkyl- phosphotriester; (iv) phosphorothioate; and (v) phosphorodithioate.
  • Non-phosphate analogs can include compounds wherein the sugar/phosphate moieties are replaced by an amide linkage, such as a 2-aminoethylglycine unit, commonly referred to as PNA.
  • Heterozygous as used herein means both members of a pair of alleles of a gene are present in a sample obtained from a single source, wherein a gene can have two alleles due to, for example, the fusion of two dissimilar gametes with respect to the gene.
  • Heterozygous assay as used herein means an assay adapted to identify the allelic state of a gene having one or both members of a pair of alleles.
  • Homozygous as used herein means one member of a pair of alleles is present in a sample obtained from a single source, wherein a gene can have one allele due to, for example, the fusion of two identical gametes with respect to the gene.
  • Homozygous assay as used herein means an assay adapted to identify only one of two possible allelic states of a gene having one or both members of a pair of alleles.
  • Lossy as used herein means the loss of haplotype diversity in a linkage disequilibrium block.
  • a library of assays can be provided.
  • the library of assays can have from about 100,000 to about 500,000 polynucleotides, for example, about 150,000 to about 250,000 polynucleotides.
  • a library of data sets can be provided.
  • the library of data sets can have from about 100,000 to about 500,000 data sets, for example, about 150,000 to about 250,000 data sets.
  • an algorithm is provided that can select a minimum subset of SNPs without loss of haplotype information, or an even smaller subset with some acceptable loss of information.
  • a SNP set was reduced by 18% for an African American population and by 32% for a Caucasian population with no loss of haplotype distribution information.
  • the algorithm can produce optimal results in a reasonable time.
  • the algorithm can allow for the real-time calculation of minimum SNP subsets for haplotype blocks.
  • an algorithm to select the minimal subset of SNPs required for capturing the diversity of haplotype blocks or other genetic loci is provided.
  • the algorithm can be used to quicldy select the minimum SNP subset with no loss of haplotype information.
  • the algorithm can be used in a more aggressive mode to further reduce the original SNP set, with minimal loss of information.
  • various embodiments can be used to eliminate additional SNPs while minimizing loss of haplotype information.
  • family relationships of the DNA donors if available, can be used to increase haplotype inference accuracy.
  • the Expectation-Maximization algorithm introduced by Excoffier and Slatkin can be accurate, especially in regions of low diversity.
  • the analysis of haplotype distributions in genetic studies aimed to find susceptibility mutations in case-control populations can be useful in finding associations. Therefore, haplotype interference can be used in disease and pharmacogenomic studies.
  • a probability vector P of length M can be defined where Pi is the relative frequency of the i ' haplotype.
  • A a haplotype/S ⁇ P allele state matrix of N columns and M rows is defined, wherein Ay (the i 1 row of the/ column of the matrix) indicates the allele state (T or '2') of the/' S ⁇ P for the t l haplotype.
  • the algorithm can eliminate columns of A while preserving as much of the information in P as possible. Quantifying the information in P can be defined using the Shannon Entropy equation:
  • the algorithm can use other measures of information.
  • the algorithm can consist of two phases (phases I and II). These phases can be performed sequentially.
  • the operations can be outlined in lossless mode as follows below.
  • any column that is identical to another column, or is the exact opposite of another column can be eliminated.
  • a column in a matrix that is identical to another column can represent a S ⁇ P that behaves identically to another S ⁇ P for all tested samples.
  • the redundant S ⁇ P will not provide any additional information.
  • a column is the exact opposite of another column in the matrix, this represents a S ⁇ P where the behavior can always be predicted from the behavior of another S ⁇ P simply by inverting it. Therefore, according to such embodiments, this S ⁇ P will not provide new information.
  • phase II it can be assumed that N columns of matrix A have been reduced to N' unique columns where N' ⁇ N.
  • any column whose elimination does not reduce the number of unique rows can be eliminated.
  • Each row in a matrix can represent the allelic states of the SNPs for a specific haplotype. Removing a "useful" SNP can eliminate the ability to detect at least one haplotype.
  • two or more haplotypes can register the same allelic state at the remaining SNPs, thereby reducing the number of unique rows. Therefore, if the elimination of a column does not reduce the number of unique rows, it can be omitted.
  • Phase I can be a "sub-set" of phase II, in the sense that if phase I is skipped, phase II can eliminate the SNPs that phase I would have eliminated. Phase I can be computationally easier to perfomi than phase II, for example, in lossy mode. Therefore it can be more efficient to begin with phase I.
  • Example 1
  • Example 1 illustrates a method according to various embodiments, in lossless mode.
  • Four SNPs and four haplotypes that yield the following allelic responses are illustrated in Table 1.
  • Haplotype 1 Haplotype 2 Haplotype 3 Haplotype 4
  • the fourth column is the exact opposite of the first column. This implies that either
  • SNP 4 or SNPi is redundant. If SNP is removed from the SNP set, no information is lost. When SNPi registers allele "1", the state of SNP 4 is known as allele "2", and conversely, when SNPi registers allele "2", the state of SNP 4 is known as allele "1". Removing SNP 4 leaves the matrix seen in Table 2.
  • Table 3 depicts the three remaining matrices, following the removal of S ⁇ Pi, S ⁇ P 2 , or SNP3, respectively.
  • the first and the third matrices only have three unique rows, whereas the second matrix has four unique rows.
  • SNP 2 can be eliminated with no loss of haplotype detection.
  • Haplotype 1 Haplotype 2 Haplotype 3 Haplotype 4
  • the set ⁇ SNPi, SNP3 ⁇ can provide the same haplotype detection ability as the full set ⁇ SNPi, SNP2, SNP3, SNP ⁇ .
  • each phase can cause the elimination of exactly one SNP.
  • each phase can result in the elimination of multiple SNPs or no SNPs.
  • the retained SNP set can be optimized to minimize the loss of haplotype detection.
  • Phase I can remain unchanged and phase II can select the optimal SNPs to eliminate.
  • the entropy H for the resulting P is computed.
  • the selection with the highest H can be chosen as the best selection.
  • N - k columns can be eliminated.
  • the resulting matrix (with k columns) can have fewer unique rows than the full matrix (with N columns).
  • the relative frequency (probability) of a "major" haplotype is equal to the sum of the frequencies of the "minor” haplotypes.
  • the repeating rows can be combined into a single row, and their respective probabilities can be summed to form a new probability.
  • the vector P can be shorter and can have larger numbers. This can reduce the value of the entropy, H.
  • the combination with the smallest reduction of entropy can be deemed the optimal selection.
  • k S ⁇ Ps can be used with no loss of information, as in Example 1.
  • Example 2 uses an LD block that was discovered using the Caucasian population panel, in Chromosome 6, overlapping the Human gene TTK (RefSeq ID ⁇ M_003318, Celera ID hCG401205) in lossy mode.
  • the block consists of 17 SNPs, and the EM algorithm inferred 8 haplotypes, with two major ones: haplotype 2 and haplotype 7 with frequencies of approximately 43% and 33%, respectively. The remaining 24% of the diversity is spread among the remaining 6 haplotypes.
  • Table 4 summarizes the allelic states of the 17 SNPs, as well as the respective probability, for each of the 8 haplotypes. Table 4.
  • the number of SNPs is reduced almost immediately to 7, with the remaining SNP set being ⁇ SNPi, SNP 2 , SNP 4 , SNPio, SNP12,
  • the optimal selection of 3 SNPs cause haplotypes 3 and 4 to merge and cause haplotypes 6, 1, and 8 to merge, with a total loss of 9.2% of original entropy.
  • the optimal single SNP is SNP ⁇ 6 . With single SNP ⁇ 6 , the detection ability is reduced to: "haplotype2" or "other.” Since haplotype 2 is the most common, with 43.2% of the frequency, if only a single SNP was chosen, SNPi ⁇ would be the most useful choice.
  • genotyping data was used from 11,160 SNPs distributed in a gene-centric fashion across chromosomes 6, 21, and 22, with intragenic spacing averaging 12 Kb, 8Kb, and 9Kb, respectively.
  • the SNPs were scored with 5'nuclease assays including TAQMAN-MGB probes from Applied Biosystems' Assays-on-DemandTM SNP Genotyping Products (Foster City, CA, USA).
  • the samples typed included 45 African-American and 45 Caucasian DNAs from the Coriell Human Diversity Collection available from Coriell Institute for Medical Research, Camden, NJ, USA.
  • LD blocks and haplotypes were computed independently for each population using methods described in Abecasis, et al., Merlin — rapid analysis of dense genetic maps using sparse gene flow trees. Nat Genet 30:97-101 (2002) and Gabriel et al., The structure of haplotype blocks in the human genome Science 296:2225-2229 (2002), both of which are herein incorporated in their entireties by reference. Only blocks of 3 or more SNPs were considered. Therefore, only 4,864 SNPs were used for the African-American population and 7,347 SNPs were used for the Caucasian population. The Caucasian population is known, in general, to have more and longer LD blocks. The algorithm was implemented in MATLAB v 6.1, available from The MathWorks Inc., Natick, MA, USA, without further optimization. The computations were completed on a 700MHz PC in less than 1 minute.
  • Table 5 summarizes the results after applying the algorithm to the haplotype blocks detected in data for chromosomes 6, 21, and 22.
  • the African- American population panel is denoted by 'A' and the Caucasian population panel is denoted by 'C.
  • Fig. 1 illustrates the relationship between the original number of SNPs in an LD block (horizontal axis) and the minimum number of SNPs required to genotype the LD block with no loss of information (vertical axis).
  • the thickness of the 'x' corresponds to the number of different blocks found in chromosome 6 with the same properties.
  • phase II can result in practically infinite execution time.
  • the largest block found in the above examples consisted of 22 SNPs.
  • comparing sub-sets of 1 to 11 out of 22 SNPs required examining over 2.4 million combinations. Even after that computation, the optimal solution is not assured since it is only a local optimum.
  • sub-sets of 12 to 22 would also need to be examined in order to assure a global optimum, bringing the total number of combinations to almost 4.2 million.
  • Various embodiments can use phase I to quickly reduce the 22 SNPs to a subset of 4 SNPs. As a result, phase II can find the global optimum (3 SNPs in lossless mode or 2 SNPs with less than 10% loss) in 15 comparisons only.
  • a counter-example is the 2 by 2 identity matrix, which is know to be full rank, but has the second column as the perfect inverse of the first column, thus providing no new information.
  • the Johnson et al. on-line supplement also provides executable programs, but the maximum subset size is set to k ⁇ 5, thereby guaranteeing suboptimal results based on the finding that the global optimum is greater than 5 in some blocks.
  • the premise of the algorithm is that the DNA sample size for each population is large enough so that the inferred haplotypes adequately represent reality. There can be a risk that a SNP whose behavior is identical to another SNP (and thus deemed worthless in terms of new information) for the sample size used, could differentiate an additional haplotype inferred with a larger sample size. This risk can be lower for common haplotypes. Rare haplotypes can harbor a causative mutation and can be present in higher frequency in some cases. Experimental errors can eliminate data points and thus can render suboptimal the minimum SNP subset. Therefore, additional SNPs can enhance the minimum SNP subset to enhance robustness.
  • databases containing information on SNPs can be used for conducting genetic studies.
  • Putative SNPs can be validated and can be assembled into a standardized SNP marker map or a database containing data sets corresponding to the standardized SNP marker map.
  • SNP information can be easily accessible and standardized assay reagents can be developed, validated, and made available to enable high throughput and automation, for example, to screen many SNPs on many individuals.
  • a reference SNP database can be produced from SNP and genomic information from both proprietary and public databases.
  • the database can be used for linkage disequilibrium (LD) mapping and can be used to provide, for example, validated, ready-to-use assays and reagents.
  • the database can provide high- density coverage of any Icnown gene regions and can enable easier and more affordable candidate gene association studies and candidate region association studies.
  • researchers can select SNPs across candidate genes or chromosomal regions that are most suitable for a given study, and can quickly translate that information into practice by, for example, directly obtaining assay protocols and reagents for those SNPs.
  • a "core" set of SNPs and the associated assay reagents can be compiled into a database or library and expanded or refined as additional information become available, such as haplotype definition for some or all of the genome.
  • the SNP database can also be used to compile a fixed set of chromosome- based assays for cost-efficient whole genome association (WGA) studies using, for example, oligonucleotide ligation assay (OLA) PCR Bead Array systems for ultra-high throughput genotyping.
  • Linkage disequilibrium is the non-random association of alleles in a chromosomal segment, and can be the basis of all genetic mapping. Selecting SNPs as genetic markers for LD studies involve considering all genetic and assay-specific technical factors that affect the ability to find association between a marker and the susceptibility mutations being mapped.
  • the extent of LD across a genomic region can dictate the SNP density necessary to ensure association between a marker and the allele sought.
  • Early attempts to model the extent of LD predicted very short LD of only a few kilobases (kb). However, recent empirical surveys report average LD levels between 5 kb and 60 kb, and extending up to hundreds of kb, which implies that the number of SNPs required for WGA studies could range from 50,000 to 250,000, and that markers spaced by tens of kb will suffice for candidate gene studies.
  • Common SNPs are the most likely to be useful for LD studies across more than one population since they represent ancient mutations that arose before ethnic group segregation. Simulation studies suggest that common SNPs are more likely than coding SNPs (cSNPs) to be in LD with a given causative allele regardless of whether the allele is present at low or high frequency.
  • common SNPs can be used to assemble a database in a hybrid gene-based approach.
  • SNPs can be considered "common" when the minor allele frequency is, for example, less than 15% in at least one of the populations used for validation.
  • a gene list can include 25,083 gene regions derived by Celera Genomics.
  • a gene region can be defined as bounded by the first and last transcribed base, including untranslated regions, plus 10 kilobases (kb) upstream and downstream to account, for example, for uncharacterized exons and regulatory regions.
  • SNPs can be selected within gene regions at an average density of, for example, one SNP per 10 kb, such that the map can resemble a gene-focused picket fence. Density for specific regions can be adjusted as data on recombination and LD extent emerges. Additional SNPs in mtergenic regions, such as, for example, non-coding regions of homology between mouse and human, can be added to a database or library.
  • obtaining a validated SNP assay at the end of a process of validating possible SNPs can be enhanced by defining prioritization criteria.
  • One criterion that can be used to validate a possible SNP is evidence of independent discovery of the minor allele.
  • a data set corresponding to a possible SNP can be cross-referenced against data available in public sources. When a data set corresponding to a possible SNP has no equivalent in the public domain, observation of the minor allele in genotypes of two independent donors can be used to validate the SNP.
  • a SNP database obtained using the above criteria can include about one million data sets corresponding to SNPs.
  • methods of confirming the existence of a SNP are provided.
  • One step can include identifying a location corresponding to a possible SNP in a polynucleotide in a first collection of data sets.
  • the first collection can contain information on genomic deoxyribonucleic acid (DNA) samples in the form of data sets corresponding to polynucleotides.
  • Another step can include confirming the existence of the SNP if at least one of a number of conditions is present or met.
  • a condition can be that a second collection of data sets containing information on genomic DNA samples contains information that identifies the location as containing the possible SNP.
  • a condition can be that 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.
  • the at least two data sets representing genomic DNA samples are obtained from two independent sources.
  • a condition can be that a data set that corresponds to a consensus sequence of genomic DNA samples in a third collection of data sets has the minor allele of the possible SNP.
  • the source of the genomic DNA of the consensus sequence and the sources of the genomic deoxyribonucleic acid (DNA) samples from the first collection of data sets are independent.
  • the third database of genomic DNA samples can be, for example, a public database of the Human Genome Project.
  • the first database of at least one genomic DNA sample can be, for example, a proprietary database of the Human Genome Project.
  • a multi-step, high-throughput assay design pipeline can be provided to ensure optimum performance of assays.
  • the methods provided can enable automation, minimize assay failure, and ensure compatibility of the SNP sequence with, for example, TAQMAN probe-based 5' nuclease chemistry, available from Applied Biosystems, Foster City, CA, and/or other assay formats.
  • a stringent scoring system can be used to select only those SNP context sequences with the highest probability of success.
  • a bioinformatics process can be used to design assays.
  • a step can involve masking SNPs adjacent to the target polymorphism and/or any sequence discrepancy between the Celera and the HGP human genome assembly, within the 600 bases of a context sequence. This can prevent primers and probes from being placed on top of other SNPs and can maximize the chance that the probes will hybridize to the correct genomic sequence.
  • TAQMAN 5' nuclease primers and probes can be designed using, for example, the ASSAYS-BY-DESIGN custom oligonucleotide reagent service (Applied Biosystems, Foster City, CA). Oligonucleotides can be designed in batch mode without manual intervention, and a scoring scheme can select the best sequences for a given SNP. The design algorithm can implement thermodynamic and heuristic rules and additional empirically-derived factors can increase manufacturability and assay performance. According to various embodiments, probes can be designed successfully for, for example, 97% of SNPs. After this step, a further computational quality-control step can also be performed in the context of the genome that can allow the elimination of potentially problematic SNP targets that may arise from repeated genomic regions, pseudo-SNPs, and/or other possible assembly artifacts.
  • primers and probes can be synthesized, and additional quality-control steps can occur.
  • oligonucleotide integrity can be tested.
  • assay performance can be tested against a panel of 10 DNA samples.
  • assays that pass post-manufacturing quality control can be validated in the population panels.
  • Assay validation in population panels can ensure that the locus is polymorphic and that the allele frequency is adequate for association studies in a variety of populations. For example, ninety (90) samples from the Coriell Human Variation Collection were obtained. By obtaining individual genotypes from a panel of 45 African Americans, a panel of 45 Caucasians, and a chimp DNA sample (to provide insight into ancestral alleles), sufficient information was obtained to estimate linkage disequilibrium between the SNPs in the LD map and to computationally infer common haplotypes. Assay validation in population panels can provide additional information on the usefulness of the markers, the coverage provided for a given study, and/or provide an independent assessment of assay performance.
  • the performance of each assay that can be comprised of at least one polynucleotide can be benchmarked against criteria, such as, for example: background signal (e.g., low signal in the control experiments run without template); signal generation (e.g., good separation between control experiments run without template and allele clusters); and specificity.
  • a criterion can be that a maximum of three clusters of fluorescing sample and a minimum of two clusters of fluorescing sample must be observed.
  • Another criterion can be that at least 90% of samples yield callable genotypes.
  • a method for compiling a library of polynucleotide data sets that each correspond to polynucleotides that 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 can include the step 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, a number of parameters are met by each polynucleotide corresponding to the data sets included in the library.
  • 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;
  • 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;
  • 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)
  • the first reaction conditions can comprise a 900 nM final primer concentration and a 250 nM final probe concentration under thermal cycling conditions.
  • the first defined value can be about 2.0
  • the second defined value can be about 1.0
  • the third defined value can be about 2.0.
  • At least, for example, 0.01% of individuals from the population can have the identifiable genotype.
  • At least 10%) of individuals from the population can have the identifiable genotype.
  • At least 20%) of individuals from the population can have the identifiable genotype.
  • the identifiable genotype can result from reacting the respective polynucleotide with the first assay reactants in the presence of the target nucleic acid sequence.
  • the reaction can occur under the first conditions.
  • the population can have a frequency of the minor allele of greater than or equal to about 5%.
  • the minor allele frequency can be greater than or equal to about 10%.
  • the minor allele frequency can be greater than or equal to about 15%.
  • methods can include not selecting a second polynucleotide data set that corresponds to a second polynucleotide if one or more of parameters (1) - (5), above, is not met by the second polynucleotide.
  • a library of polynucleotide data sets can be compiled using methods according to various embodiments.
  • a library of assays can be compiled using methods according to various embodiments.
  • the method can include manufacturing a library of assays wherein each assay can be made using a polynucleotide data set compiled in the library.
  • a library of polynucleotides can be compiled by manufacturing polynucleotides corresponding to polynucleotide data sets compiled using methods according to various embodiments.
  • a library of assays can be compiled using methods according to various embodiments. According to various embodiments, a method of detecting a SNP can be provided.
  • a step of the method can be reacting a sample containing a target nucleic acid sequence that has a target SNP with an assay selected from the library of assays compiled according methods described herein.
  • a step can be determining the genotype of the target nucleic acid sequence that has the target SNP by detecting a characteristic attributable to the genotype of the target SNP in the sample.
  • a method for 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 can include the step of 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.
  • the method can include the step of 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.
  • the method can include the step of 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.
  • the method can include the step of 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.
  • the method can include the step of 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.
  • the method can include a combination of some of or all of these steps.
  • the polynucleotide data set corresponding to the respective polynucleotide can be selected for the library if, for example, the background signal value in parameter (1) is less than or equal to about two, if the ratio from the comparison in parameter (2) is greater than or equal to about one, if the ratio from the comparison in parameter (3) is less than or equal to about two, if the at least one individual of parameter (4) has the identifiable genotype, and if the at least one individual of parameter (5) has the identifiable minor allele.
  • a library of polynucleotide data sets can be compiled using methods according to various embodiments.
  • a library of polynucleotides can be compiled by manufacturing polynucleotides corresponding to polynucleotide data sets compiled using a method or methods according to various embodiments.
  • a method of compiling a library of assays can be provided.
  • the method can include manufacturing a library of assays, wherein each assay is manufactured using a polynucleotide data set compiled in a library according to various embodiments.
  • methods of detecting a SNP can be provided.
  • the method can include the step of reacting a sample containing a target nucleic acid sequence that has a target SNP with an assay selected from the library of assays compiled using a method or methods according to various embodiments.
  • a step can include determining the genotype of the target nucleic acid sequence that has the target SNP by detecting a characteristic attributable to the genotype of the target SNP in the sample.
  • an automatic allele calling software can be used to automatically analyze validated assay data without user intervention. According to various embodiments, at least, for example, 90% of the assay data can be processed automatically to identify an allele. According to various embodiments, an automated validation process can be used for high volume commercial or research purposes.
  • Fig. 4 is a plot 100 of fluorescence data from many SNP assays according to various embodiments.
  • the x-axis represents relative fluorescence of a 6-FAM dye label and the y- axis represents relative fluorescence of a VIC dye label.
  • Cluster 110 represents the relative fluorescence of control samples having probes labeled with 6-FAM and VIC, respectively. The control samples did not contain a target nucleic acid sequence.
  • the background signal value is the average of the relative fluorescence of the control samples as represented by cluster 110 in Fig. 4.
  • the background signal value in Fig. 4 is less than about 2.0.
  • Cluster 120 represents the relative fluorescence of samples having homozygous alleles (allele 2) that hybridized with probes labeled with 6-FAM.
  • Cluster 130 represents the relative fluorescence of samples having heterozygous alleles (alleles 1 and 2) that hybridized with probes labeled with VIC and 6-FAM, respectively.
  • Cluster 140 represents the relative fluorescence of samples having homozygous alleles (allele 1) that hybridized with probes labeled with VIC.
  • the signal generation value for the assays is based, at least in part, on the average of the relative fluorescence of at least one of clusters 120, 130, and 140.
  • Fig. 5 is a flowchart showing a comparison of reference values determined by experimental assays according to various embodiments against target values.
  • TBV is a target background value
  • TsigV is the target signal value
  • TSpV is the target specificity value
  • TIP is the target identifiable percentage, or the minimum frequency that the assay produces an identifiable genotype
  • TMI is the target minor allele frequency, or the minimum frequency that the minor allele appears in the population.
  • Fig. 6 is a flowchart that illustrates the selection of a data set into a library of data sets, according to various embodiments.
  • Experimental or reference data is obtained by performing an assay against individuals in a population. Experimental data can be obtained at least until results are statistically significant for a given population. When a statistically significant sample size has been determined, the experimental data can be processed according to various embodiments as shown, for example, in Fig. 5. If the selection criteria are met, the data set corresponding to polynucleotides used in the experimental assays can be added into the library. If the selection criteria are not met, the polynucleotide can be redesigned or discarded, where the expendable assays can be repeated.
  • SNP assays and reagents can use easily automated chemistry and can be compatible with readily available high-throughput instrumentation and software systems.
  • SNP assays and reagents can have few enzymatic steps, no post-reaction transfer of liquids, and/or universal reaction conditions that can facilitate robotic liquid handling automation.
  • the assays, reagents, and/or high-throughput workflow can be easy to implement and automate and/or can use components that are ready-to-use out-of-the-box and require no optimization.
  • TAQMAN probe-based 5' nuclease assay chemistry available from Applied
  • Biosystems, Foster City, CA can meet almost any assay requirement and can unite PCR amplification and signal generation into a single step, thereby simplifying automation of both reaction set-up and data collection.
  • a hybridization probe with fluorogenic and quencher tags is cleaved by the 5' nuclease activity of thermus aquaticus (Taq) DNA polymerase during PCR amplification. Cleavage produces fluorescence by freeing the fluorogenic molecule from the quencher.
  • Taq thermus aquaticus
  • Figs. 2a-2e provide an overview of the TAQMAN probe-based 5' nuclease assay chemistry for SNP genotyping.
  • the TAQMAN system is adapted to provide allelic discrimination and high- throughput SNP genotyping.
  • Chemistry improvements have increased assay design flexibility, enabled easy protocol standardization, enabled the use of universal reagents, and reduced background fluorescence, all of which can be desirable for high throughput SNP processing and allelic discrimination.
  • conjugated probes can significantly stabilize probe-template complexes, enabling the use of probes in the 13-mer to 20-mer size range.
  • conjugated probes can have better mismatch discrimination, can be easier to design for challenging genetic regions such as those high in GC content or those in variable context sequences, and/or can increase the signal-to-noise ratio by bringing the quencher closer to the fluorescent tag.
  • conjugated probes can increase the melting temperature window that can be used in reaction protocols, thereby allowing all the SNP assays to run under identical conditions.
  • Previous 5' nuclease quenchers emitted their own fluorescence, therefore signal detection was complicated. New non-fluorescent quenchers can provide improved signal detection and can facilitate automated allele calling, another desirable feature useful for high-throughput SNP scoring.
  • the most precious reagent can be the DNA sample itself.
  • the 7900HT system can use a 5 microliter reaction volume and can consume one nanogram of DNA per genotyping reaction, thereby minimizing reagent costs and conserving DNA template samples.
  • the 5' nuclease assay can be suited for automation because of its easy, three-step workflow.
  • a universal master mix including probes and primers, can be added directly to plates of dry or fresh DNA using standard robotics. Plates can be sealed and cycled using standard thermal cyclers, such as, for example, the Applied Biosystems Dual 394- Well GENEAMP PCR System 9700 thermal cycler, available from Applied Biosystems, Foster City, CA. Following cycling, plates can be automatically read on the 7900HT that can support the collection of more than 250,000 genotypes per day.
  • the availability of thermal cyclers with automated lid handling can increase throughput by enabling robotics integration for 24-hour unattended operation. Automation software can also increase both quality and throughput. For example, automation of allele calling can remove inter- technician variability, increasing confidence in data quality and reducing the time spent on data analysis by 8.5 person-hours per day.
  • Fig. 3 provides an example of an automated workflow system.
  • an assay that uses two different types of probes can be provided wherein the polynucleotide and the reporter dyes differ.
  • the first type of probe can have a first polynucleotide with a VIC reporter dye attached to the 5' end of the first polynucleotide
  • the second type of probe can have a second polynucleotide with a 6-FAM reporter dye attached to the 5' end of the second polynucleotide
  • the first and second polynucleotides can differ by at least one nucleic acid residue at the same location in the polynucleotide when the polynucleotides are aligned 5' to 3'.
  • the dye-labeled probes can be adopted to perform a heterozygous assay or a homozygous assay.
  • the probe can anneal to a complementary sequence between the forward and reverse primer sites. At the time of annealing, the probe is intact and the proximity of the reporter dye to the quencher can result in suppression of fluorescence of the reporter dye.
  • a polymerase can cleave a reporter dye only when the probe has completely, mostly, or substantially hybridized to the target DNA sequence. When the reporter dye is cleaved from the probe, the relative flouorescence of the reporter dye increases. The increase in relative fluorescence can be caused to only occur if the amplified target DNA sequence is complementary, mostly complementary, or substantially complementary to the probe. Therefore, the fluorescent signal generated by PCR amplification can indicate which alleles are present in a sample.
  • Mismatches between a probe and a target DNA sequence can reduce efficiency of probe hybridization and/or a polymerase can be more likely to displace a mismatched probe without cleaving it and therefore not produce a fluorescent signal. For example, if one of two possible reporter dyes fluoresce during an assay, then the presence of a homozygous gene is indicated. For further example, if both possible reporter dyes fluoresce during an assay, then the presence of a heterozgous gene is indicated.
  • At least one primer can be provided, wherein the primer can be a sequence that is shorter than the target DNA sequence.
  • the primer can have a polynucleotide and/or a minor groove binder.
  • the primer can be a sequence that is complimentary to, or mostly complimentary to, the target DNA sequence.
  • the primer can be at least 90% homologous to a corresponding length of the target DNA sequence, at least 80%) homologous to a corresponding length of the target DNA sequence, at least 70% homologous to a corresponding length of the target DNA sequence, or at least 50% homologous to a corresponding length of the target DNA sequence.
  • thermostable DNA polymerase such as, for example, thermus aquaticus (Taq), and at least 4 embodiments of a deoxyribonucleic acid (e.g., adenosine, tyrosine, cytosine, and guanine)
  • the polymerase can be, for example, AMPLITAQ GOLD, available from Applied Biosystems, Foster City, CA.
  • components of a fluorogenic 5' nuclease assay or other assay reagents that utilize 5' nuclease chemistry for example, TAQMAN minor groove binder probes, available from Applied Biosystems, Foster City, CA, can be provided.
  • Some or all of the above-listed components can be replaced by or used with commercially- available products, for example, buffers or AMPLITAQ GOLD PCR MASTER MIX (Applied Biosystems, Foster City, CA).
  • a high-quality LD map of validated SNPs was created by integrating information from both public and private human genome efforts.
  • a set of over 200,000 validated, easy-to-use, individual SNP assays and TAQMAN ready-to-use assay reagents created by using methods according to various embodiments can be provided.
  • a minor groove binder and a non-fluorescent quencher, and the integration of the 5' nuclease chemistry with an automated detection system, such as, for example, the 7900HT, can be used.
  • a web-based bioinformatics and ordering system can be provided where a customer can search for SNPs and order assay reagents, thus reducing the time and costs associated with candidate-gene and candidate-region association studies.
  • and LD map can, for example, enable candidate-gene and candidate-region association studies using 5' nuclease chemistry and/or be implemented on an ultra-high throughput SNP genotyping platform to enable WGA studies.
  • the 5' nuclease chemistry system can leverage the specificity of the OLA-PCR assay chemistry and the highly parallel detection of, for example, BEAD ARRAY technology, available from Illumina, Inc., San Diego, CA.
  • the system can enable the generation of about 2,100,000 genotypes per day and all components of the assay can be universal except for, for example, the SNP-specific OLA probes.
  • assays for over 4,000,000 SNPs from the Celera database can cover every gene in the human genome. According to various embodiments, many SNPs can have the necessary variability for genetic association studies and assays for the SNPs can be provided. According to various embodiments, assays can be grouped together into convenient SNP sets optimized for specific assays such as, for example, p450 genotyping and disease-specific gene studies.
  • Figs. 2a-2e are schematic diagrams showing the interaction of components that can be part of a mixture of reagents according to various embodiments, hi Fig. 2a, primer 52 has annealed to template strand 54. Replication of the template strand from primer 52 will occur in the 5' to 3' direction.
  • Probe 50 including a generic reporter dye R, quencher Q, and minor groove binder MGB, has annealed to the template strand 54.
  • Arrow 53 shows that as the complementary strand (not shown) is produced from the template strand 54 starting at the forward primer 52, the complementary strand will meet probe 50.
  • Fig. 2b shows the 5 complementary strand 55 as it meets probe 50a.
  • Polymerase 60 cleaves VIC reporter dye V during the production of complementary strand 55 given that probe 50a has annealed to the target strand 54 because the target strand 54 and the probe 50a are completely complementary.
  • Fig. 2c shows the complementary strand 55 as it meets probe 50b.
  • Polymerase 60 does not cleave FAM reporter dye F during the production of complementary strand 55 given that probe 0 50b has not hybridized with the target strand 54 because of a mismatched base pair at location 64.
  • Fig. 2d shows the complementary strand 55 as it meets probe 50b.
  • Polymerase 60 cleaves FAM reporter dye F during the production of complementary strand 55 given that probe 50b has annealed to the target strand 54 because the target strand 54 and the probe 50b are completely complementary.
  • Fig. 2e shows the complementary strand 55 as it meets probe 5 50a.
  • Polymerase 60 does not cleave VIC reporter dye V during the production of complementary strand 55 given that the probe 50a has not hybridized with the target strand 54 because of a mismatched base pair at location 66.
  • Fig. 7 is an illustration of SNPs selected by a method according to various embodiments.
  • a library is provided that contains a plurality of data sets, corresponding to one or more respective oligonucleotides that can function as a respective assay to hybridize with at least one respective Single Nucleotide Polymorphism (SNP) in a nucleic acid sequence.
  • the nucleic acid sequence can include three or more adjacent SNPs and the data sets can correspond to at least the three or more adjacent SNPs, 5 respectively.
  • Each adjacent SNP can be spaced a distance from at least one other adjacent SNP and each of the distances between adjacent SNPs can be from about 75% to about 125% of an average of the distances between the adjacent SNPs.
  • the corresponding, adjacent SNPs can be spaced apart along at least a region of a chromosome.
  • the corresponding, adjacent SNPs can be spaced apart along at least a region of a gene.
  • the 0 distances between all corresponding, adjacent SNPs can be equal, plus or minus 30%.
  • the distances between all corresponding, adjacent SNPs can be equal, plus or minus 20%.
  • Each of the distances between corresponding, adjacent SNPs can be from about 90% to about 110%) of an average of the distances between the adjacent SNPs.
  • Each of the distances between corresponding, adjacent SNPs can be from about 95% to about 105% of an average of the distances between the adjacent SNPs.
  • the nucleic acid sequence can be a consensus sequence.
  • the distances between all corresponding, adjacent SNPs can be less than a specified maximum distance.
  • the specified maximum distance can be, for example, 10 kilobases, 15 kilobases, 20 kilobases, or 30 kilobases.
  • the algorithm can select a minimum number of SNPs per region. For example, three SNPs per gene can be selected.
  • the distances between all corresponding, adjacent SNPs can be greater than a specified minimum distance.
  • the nucleic acid sequence can be a consensus sequence corresponding to the human genome.
  • the nucleic acid sequence can be a nucleic acid sequence data set.
  • the library can comprise a number of data sets corresponding to not more than a sufficient number of oligonucleotides necessary to provide a collection of assays that can provide a maximum statistical loss of haplotype diversity, across the region, of less than ten (10) percent.
  • the sufficient number of oligonucleotides can be obtained by providing a matrix comprised of data representing haplotype blocks and SNP locations.
  • the columns of the matrix can contain data representing existence of respective SNPs within a haplotype block and the rows of the matrix can contain data representing respective haplotype blocks.
  • At least one column can be eliminated, wherein elimination of the at least one column may not reduce the number of rows in the matrix that contains non-duplicative information.
  • At least one column of the matrix that is identical to a second column of the matrix and/or completely opposite to a second column of the matrix can be eliminated.
  • Fig. 7 illustrates SNPs that can be selected according to various embodiments.
  • SNPs 710, 720, 730, and 740 that are present in region 702 of nucleic acid sequence 700 have been selected based on prioritization criteria.
  • SNPs 710, 720, 730, and 740 are separated from adjacent SNPs by a distance no greater than distance 760.
  • the effective range of usefulness of each SNP is equal to plus or minus one half of distance 760. Therefore, there are no gaps in coverage of region 702 of nucleic acid sequence 700.
  • SNPs 750, 752, 754, 756 were not selected using the same selection criteria.
  • the selection criteria can be used to select data sets for the library, where the data sets correspond to one or more respective oligonucleotides that can function as a respective assay to hybridize with at least one respective SNP.
  • at least one selection criterion of the selection criteria can be used.
  • One criterion can be a specified maximum distance between adjacent SNPs.
  • Another criterion can be a specified minimum distance between adjacent SNPs.
  • a criterion can be a target distance between adjacent SNPs. The actual distance between adjacent SNPs can vary from the target distance by, for example, 5%, 10%, 20%, or 30%.
  • An algorithm to prioritize SNPs can select as few SNPs as possible to achieve a coverage of the region where the largest gap between adjacent SNPs is less than or equal to the specified maximum distance.
  • An algorithm to prioritize SNPs may not select a SNP if the distance between one of two adjacent SNPs is less than the specified minimum distance.
  • Another prioritization criterion can involve using known SNPs.
  • Known SNPs can be preferentially selected to include in the library because, for example, Icnown SNPs are well-characterized, and may have assays that have previously been designed to target such known SNPs, they may have little or no additional cost associated with providing an oligonucleotide or a data set corresponding to an oligonucleotide for an assay directed to the SNP.
  • An algorithm to prioritize SNPs can consider known SNPs as "must use” SNPs or as "prefer to use” SNPs.
  • a Icnown SNP is a previously characterized SNP marker that has low or no cost associated with designing and / or manufacturing one or more oligonucleotides.
  • Known SNPs can be preferentially used as a selection criterion.
  • a newly identified SNP is a SNP marker that is relatively uncharacterized and therefore there is a higher relative cost associated with designing and / or manufacturing one or more oligonucleotides.
  • a selection criterion can be newly identified SNP.
  • Newly identified SNPs can be selected after all or some of the known SNPs have been selected because, for example, newly identified SNPs require investigation into whether a functional assays can be produced for the newly identified SNPs and therefore newly identified SNPs have greater costs associated with them than Icnown SNPs.
  • An algorithm to prioritize SNPs can consider newly identified SNPs as “must never use” SNPs, "use only after using all known” SNPs, or as "try not to use” SNPs.
  • selection criteria can be used to select data sets corresponding to the largest number of known SNPs and the smallest number of newly identified SNPs that provide a coverage of the region, where the distance between adjacent, selected SNPs is approximately equal.
  • a specified maximum distance can be allowed between two adjacent selected SNPs, as well as between the two outermost selected SNPs and the boundaries of the region, gene, chromosome, or other boundary denoting the beginning and ending of the nucleic acid sequence.
  • One requirement can be, for example, to never have a distance between adjacent selected SNPs that is greater than a maximum required distance, unless maintaining the maximum required distance is impossible because there are no adjacent SNPs that fall within the maximum required distance.
  • the largest distance between adjacent selected SNPs can be as small as possible.
  • an algorithm can be provided that selects, based on prioritization criteria, a plurality of data sets that corresponds to one or more respective oligonucleotides that can function as a respective assay to hybridize with at least one selected SNP.
  • a region can be at least a part of the nucleic acid sequence.
  • the region can be separated into sub-regions.
  • the sub-regions can, for example, be separated by known SNPs, if any.
  • Each sub-region can be solved and optimized independently according to various embodiments of the algorithm.
  • the sub-region can be bound by known SNPs. If there are no known SNPs, then the region may not be separated into sub-regions.
  • Various embodiments of the algorithm can be repeated for each sub-region.
  • Each sub-region can begin, for example, with the region's 5' end or a Icnown SNP and can end with the 3' end or a known SNP.
  • a step of the algorithm can be a locally optimal solution (“local step") that can determine the number of newly identified SNPs that can be utilized.
  • the step can include selecting all the newly identified SNPs.
  • the step can include selecting the known SNPs closest to one or both ends of the region or sub-region of the nucleic acid sequence.
  • the step can include selecting the newly identified SNPs closest to one or both ends of the region or sub-region of the nucleic acid sequence.
  • the step can iteratively eliminate newly identified SNPs until, for example, no newly identified SNPs can be eliminated.
  • the step can iteratively eliminate newly identified SNPs until, for example, the remaining number of Icnown SNPs and newly identified SNPs is at or below a minimum number of SNPs, if any, that can be selected.
  • the distance between all adjacent SNPs can be calculated.
  • the first and/or last distances between adjacent SNPs in a region or sub-region can be doubled if the distances are between a SNP and the edge of the region or sub-region. From the 5' end of the region or sub-region, the consecutive distances between adjacent SNPs can be added until the cumulative sum of consecutive distances exceeds the specified maximum distance.
  • the local step can include identifying the smallest distance between adjacent SNPs in the sequence of adjacent SNPs that makes up the cumulative sum, where the cumulative sum of adjacent distances is greater than the specified maximum distance. If there is only one distance between adjacent SNPs having a distance equal to the smallest distance, then one of the two adjacent SNPs bounding the smallest distance can be eliminated if one of the SNPs is a newly identified SNP.
  • each distance value is the distance between two adjacent SNPs
  • each SNP can have two distance values associated with it.
  • the distance value (5') is the distance to the adjacent SNP on the 5' end of the nucleic acid sequence
  • the distance value (3') is the distance to the adjacent SNP on the 3' end. If there is more than one distance between adjacent SNPs having a distance equal to the smallest distance, e.g.
  • the tie can be broken by, for example, choosing the smallest distance on the 3' end of the nucleic acid sequence.
  • the smallest distance can be chosen arbitrarily (e.g. the smallest distance closest to the 5' end, the smallest distance closest to the 3' end, or the smallest distance that falls in the middle of the other smallest distances).
  • the cumulative sum can be recomputed and the process can be reiterated using the remaining SNPs. According to various embodiments, if no newly identified SNPs can be removed, the process of the local step can be stopped.
  • a step of the algorithm can be a globally optimal solution (“global step") that can determine the optimum selection of newly identified SNPs given the number of newly identified SNPs that can be utilized. For example, the number of newly identified SNPs that can be utilized can be provided by the local step. According to various embodiments, at least one step of the algorithm is performed.
  • N can be a value from 1 to K.
  • K can be selected by determining the specified minimum and/or specified maximum distances of the region or sub-region.
  • the global step can include calculating all possible selections P of the number of newly identified SNPs to be selected N out of the total number of newly identified SNPs in the region or sub-region K.
  • the global step can include calculating the largest distance between adjacent, selected SNPs for each selection P.
  • the global step can include choosing the selection P with the "smallest" largest distance between adjacent, selected SNPs.
  • the global step can be illustrated by the following operation:
  • N The smallest value of N can be selected where Min ⁇ Max [SNPi - SNPi-i] ⁇ (where i- 1 , ... ,N), that is less than T.
  • a specified minimum distance between adjacent selected SNPs can be specified.
  • a minimum number of total markers can be specified.
  • Prioritization criterion can be assigned to different, respective newly identified SNPs that can assign preference to some newly identified SNPs over other newly identified SNPs.
  • the distance value associated with a "high priority" newly identified SNP can be, for example, marked or changed so that the "high priority" newly identified SNP is always preferred or selected over a "low priority" newly identified SNP.
  • Fig. 8 details a hypothetical chromosome having 969 genes on the chromosome. Of those 969 genes, 1,639 Icnown SNPs are present on the chromosome and are well characterized.
  • the chromosome contains 11,095 newly identified SNPs that are not well characterized. Of the 969 genes, 611 genes contain SNPs and 358 genes do not contain SNPs. Of the 611 genes containing SNPs, Fig. 8 lists the average gene length, in bases, the number of newly identified SNPs per gene, the number of Icnown SNPs per gene, the total number of selected SNPs per gene that were selected using various embodiments, and the number of newly identified selected SNPs per gene that were selected using various embodiments.
  • Fig. 9 is a histogram of gene lengths of the genes found on the hypothetical chromosome of Fig. 8. Fig.
  • Fig. 10 is a histogram of the specified maximum distance between adjacent SNPs, according to various embodiments, of the selected SNPs from the hypothetical chromosome of Fig. 8.
  • Fig. 11 is a histogram of the actual maximum distance between adjacent SNPs, according to various embodiments, of selected SNPs of the hypothetical chromosome of Fig. 8.
  • Fig. 12 is a histogram of total selected SNPs per gene, according to various embodiments, from the hypothetical chromosome of Fig. 8.
  • Fig. 13 is a histogram of the number of newly identified SNPs per gene that were selected from the hypothetical chromosome of Fig. 8 using various embodiments.

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Abstract

L'invention concerne des banques de dosages et des procédés de compilation desdites banques. Lesdits dosages peuvent identifier des polymorphismes nucléotidiques simples (SNP). L'invention concerne également des procédés de validation des SNP. L'invention concerne en outre des procédés de construction de cartes de déséquilibre de liaison au moyen d'ensembles ou de sous-ensembles de SNP.
EP03707532A 2002-01-25 2003-01-27 Procedes de validation de snp et de compilation de banques de dosages Withdrawn EP1483405A4 (fr)

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US30012803P 2003-01-02 2003-01-02
US10/335,690 US20040063109A2 (en) 2002-01-25 2003-01-02 Single-tube, ready-to-use assay kits, and methods using same
US10/335,707 US20030190652A1 (en) 2002-01-25 2003-01-02 Methods of validating SNPs and compiling libraries of assays
US300128P 2003-01-02
US335690 2003-01-02
PCT/US2003/000128 WO2003065146A2 (fr) 2002-01-25 2003-01-02 Procedes permettant de placer, d'accepter et de classer des commandes relatives a des produits et des services
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US10/334,793 US20040018506A1 (en) 2002-01-25 2003-01-02 Methods for placing, accepting, and filling orders for products and services
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Citations (2)

* Cited by examiner, † Cited by third party
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EP0892068A1 (fr) * 1997-07-18 1999-01-20 Genset Sa Méthode pour la génération d'une carte du génome humain avec haute densité basée sur linkage diséquilibrium
US6100030A (en) * 1997-01-10 2000-08-08 Pioneer Hi-Bred International, Inc. Use of selective DNA fragment amplification products for hybridization-based genetic fingerprinting, marker assisted selection, and high-throughput screening

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Publication number Priority date Publication date Assignee Title
US6100030A (en) * 1997-01-10 2000-08-08 Pioneer Hi-Bred International, Inc. Use of selective DNA fragment amplification products for hybridization-based genetic fingerprinting, marker assisted selection, and high-throughput screening
EP0892068A1 (fr) * 1997-07-18 1999-01-20 Genset Sa Méthode pour la génération d'une carte du génome humain avec haute densité basée sur linkage diséquilibrium
WO1999004038A2 (fr) * 1997-07-18 1999-01-28 Genset Marqueurs bialleles convenant a la constitution d'une carte haute densite des desequilibres du genome humain

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