Automated analysis of multiplexed probe-target interaction patterns: pattern matching and allele identification

Abstract

Disclosed are methods and algorithms (and their implementation) supporting the automated analysis and interactive review and refinement (“redaction”) of the analysis within an integrated software environment, for automated allele assignments. The implementation, preferably with a software system and a program referred to as the Automated Allele Assignment (“AAA”) program, provides a multiplicity of functionalities including: data management by way of an integrated interface to a portable database to permit visualizing, importing, exporting and creating customizable summary reports; system configuration (“Set-up”) including user authorization, training set analysis and probe masking; Pattern Analysis including string matching and probe flipping; and Interactive Redaction combining real-time database computations and “cut-and-paste” editing, generating “warning” statements and supporting annotation. It also includes a thresholding function, a method of setting thresholds, a method of refining thresholds by matching an experimental binary string (“reaction pattern”) setting for that probe, probe masking of signals produced by probes which do not contribute significantly to discriminating among alleles.

1 - 28 . (canceled) 
     
     
         29 . A method for reducing erroneous allele assignments where assignment is made based on the results of a hybridization assay or capture-mediated elongation assay and multiple polymorphic loci are present on each allele, comprising:
 (i) selecting a set of primers for generating targets from genomic regions which include the polymorphic loci of interest;   (ii) selecting a set of probes complementary, in whole or in part, to subsequences of the targets;   (iii)(a) determining potential ambiguity, i.e., if positive assay results from the selected set of probes can generate a probe-subsequence interaction reaction pattern representative of more than one allele or more than one pair of two or more known allele pairs (which could be present in a sample from a subject), and:   (iii)(b) if there is no potential ambiguity, selecting the probe set for analysis of samples and not proceeding to step (iv) or (v);   (iii)(c) if there is potential ambiguity, deleting the probes associated with the potential ambiguity from the probe set and selecting different probes in step (ii) and repeating step (iii)(a) as many times as needed to attempt to eliminate the ambiguity, and if the ambiguity can be eliminated following several iterations of step (iii)(a), selecting the probe set for analysis of samples and not proceeding to step (iv) or (v); but if the ambiguity cannot be eliminated;   (iv) repeating step (i) to (iii) but selecting a new set of primers including at least some primers having different sequences than the set previously used in step (i); and   (v) repeating step (iv) several times to attempt to eliminate the ambiguity.   
     
     
         30 . The method of  claim 29  wherein the hybridization assay is positive when hybridization takes place. 
     
     
         31 . The method of  claim 29  wherein the capture-mediated elongation assay is positive when elongation takes place. 
     
     
         32 . The method of  claim 29  further including, using the selected probe and primer sets to perform a simulated multiplexed hybridization reaction between the selected probes and their targets at a specified annealing temperature consistent with the expected annealing temperatures of as many of the probes pairing with their complementary subsequences in the targets as possible, but for those pairings which have annealing temperatures so significantly below the specified annealing temperature that no significant degree of annealing is expected to take place at the specified temperature, the corresponding probes are deleted from the probe set and steps (ii) and (iii) of  claim 29  are repeated; and, optionally, if suitable probes cannot be selected after several repetitions of steps (ii) and (iii), steps (iv) and (v) of  claim 29  are performed. 
     
     
         33 . The method of  claim 29  further including labeling some targets generated by the selected primer set differently from others. 
     
     
         34 . The method of  claim 33  wherein a set of targets are determined which present a high potential to generate ambiguities, and some members of said set are labeled differently from others in a manner so as to reduce said potential. 
     
     
         35 . The method of  claim 34  wherein the high potential to generate ambiguities results from a single nucleotide polymorphism in the allele which aligns with the 3′ terminus of the primer. 
     
     
         36 . The method of  claim 29  wherein following step (i), known alleles which include the polymorphic loci of interest are aligned to more accurately identify subsequences of polymorphic loci suitable for primer amplification and targeting with probes. 
     
     
         37 . A probe set produced by the methods of  claim 29 . 
     
     
         38 - 44 . (canceled) 
     
     
         45 . A method for establishing a series of thresholds to be used for reducing erroneous allele assignments, where assignment is made based on the results of a hybridization assay or capture-mediated elongation and multiple polymorphic loci are present on each selected allele in a sample, comprising:
 (i) selecting a reference set of targets derived from a sample, where subsequences in the targets have a known reaction pattern with a selected set of probes, and known allele assignments;   (ii) reacting the set of targets with the selected set of probes to obtain a pattern representing the matching subsequences and probes of varying intensities, where each intensity which is greater than a pre-determined threshold is designated as a positive reactions between a probe and a subsequence;   (iii) subtracting from each signal intensity, I, the negative control signal, I NC , and dividing the result by the corrected positive control signal, I PC , to obtain a normalized intensity (ratio):
     r =( I−I   NC )/( I   PC   −I   NC ); and 
   (iv) determining, for each probe, k, in the selected set, a threshold, T k  for each r k , where values of r k  above the threshold are designated as positive for the corresponding probe-subsequence interaction and values of r k  below the threshold are designated as negative for the corresponding probe-subsequence interaction, and where T k  is determined so as to maximize the match between the actual reaction pattern of the set of targets with the probes in the set, and the known reaction pattern for said set of targets with the probes in the set.   
     
     
         46 . The method of  claim 45  further including the step of using the thresholds to determine positive and negative probe-subsequence interactions in a different sample, and make allele assignments for said sample. 
     
     
         47 . The method of  claim 46  wherein allele assignments are only made following a comparison of the reaction pattern with the predicted reaction pattern from a reference for known alleles. 
     
     
         48 . The method of  claim 47  wherein following the comparison, certain probe-subsequence interaction intensity results which do not correlate with the predicted reaction pattern for the alleles the targets were derived from, are inverted, i.e., negative is made positive or positive is made negative. 
     
     
         49 . The method of  claim 48  wherein the inversions are made to correlate as closely as possible with a predicted reaction pattern for a known allele. 
     
     
         50 . The method of  claim 47  wherein the allele assignments are made to correlate as closely as possible with a predicted reaction pattern. 
     
     
         51 . The method of  claim 47  wherein samples are derived from a subpopulation with a known allele distribution, and the predicted reaction pattern is for said subpopulation. 
     
     
         52 . The method of  claim 45  wherein the actual reaction pattern is analyzed to determine if the threshold is representative of the difference between samples generating two reactive subsequences for a probe and those generating only one reactive subsequence, or whether the threshold is representative of the difference between samples generating one reactive subsequence for a probe and those generating no reactive subsequences. 
     
     
         53 . The method of  claim 47  wherein allele assignments which do not correlate with a predicted reaction pattern are analyzed and an assignment is made considering the predicted reaction pattern and the possibility of a new allele. 
     
     
         54 . The method of  claim 45  further including the step of determining the goodness, G, of a threshold setting for a particular probe determined in assays against a set of two or more different samples, where:
     G =( C   1   +C   2 )/(2* C   0 ), 
 
       and where C 0  denotes the maximum value of C 1  (the goodness of one probe in the selected set) for the set of samples, C 1  is the value of C 1  when the threshold value increases by a certain percentage, and C 2  is the value of C 1  when the threshold value decreases by a certain percentage.