Patent Application: US-201514937228-A

Abstract:
a total forensic dna casework management system and method for the deconvolution of mixed dna samples using a novel , 3 - rule algorithm to determine the proportional allele sharing of the sample &# 39 ; s contributors . the process is fully document , can assess and process dna anomalies and artifacts , and transforms raw str data to produce final dna profile types , peak height ratios , proportions , fitting criteria and associated graphs .

Description:
the practice of the present invention will employ , unless otherwise indicated , conventional methods of chemistry , biochemistry , recombinant dna techniques and immunology , within the skill of the art . all publications , patents and patent applications cited herein , whether supra or infra , are hereby incorporated by reference in their entirety . it must be noted that , as used in this specification and the appended claims , the singular forms “ a ”, “ an ” and “ the ” include plural referents unless the content clearly dictates otherwise . thus , for example , reference to an antigen includes a mixture of two or more antigens , and the like . in describing the present invention , the following terms will be employed , and are intended to be defined as indicated below unless otherwise noted : the interpretation of mixtures requires the understanding of at least two pcr phenomena assumed to be the result of stochastic variation in the amplification process or sampling of template : heterozygote balance ( hb ) and variation in mixture proportion ( mx ). in addition we assume that peak area is approximately linearly proportional to the amount of dna prior to amplification and that contributions from two separate alleles are additive . heterozygous balance ( hb ) describes the area ( or height ) difference between the two peaks of a heterozygote . this has been previously defined in two different ways either as the ratio of the smaller area peak to the larger area peak [ 13 ]: or as the ratio of the high molecular weight ( hmw ) peak to the lower ( lmw ): it can be shown , using artificial mixtures , that peak areas corresponding to an allelic position are approximately proportional to the amount of dna from the contributor however , this proportionality is imprecise and is affected by many factors such as locus ; degradation ; the presence of stutter ; stochastic variation and other artifacts , especially when the concentration of dna is low . allele drop - in : contamination from a source unassociated with the crime stain manifested as one or two alleles . allele drop - out : low level of dna insufficiently amplified to give a detectable signal . artifact peaks are peaks due to impurities in the dna samples . generally , the artifact peaks have one or more of the following three characteristics : ( 1 ) about 53 % of them are less than 5 % of the nearest allele peak &# 39 ; s height , ( 2 ) some artifact peaks consist of multiple peaks , and the distances among them are always less than 1 bp , and ; ( 3 ) some artifact peaks are within 0 . 5 bp of an allelic ladder marker . if a peak satisfies any of the above three rules , the peak can be defined as an artifact peak , and the peak &# 39 ; s effect can be eliminated . “ best - fit ” refers to an assumption that the allele peak area / height is proportional to the relative mass proportion of the corresponding dna allele in the mixture , the returned genotypes at the specified mass proportions would yield a set of allele peak areas / heights that is ‘ closest ’ to the measured set of allele areas / heights , in the least square sense ( as measured by the euclidean distance metric ). conservative : 1 . an assignment for the weight of evidence that is believed to favor the defense ; or , 2 . when the evidence is very powerful in one direction , assigning the weight as less than our belief in that direction ; or , 3 . lack of conservativeness will often result when the assumptions that underpin a statistical model are seriously violated . contamination : extraneous dna from a source unassociated with the crime stain — e . g . plastic - ware can be contaminated at manufacturing source . continuous approach : the allelic intensity information is used to give a variable , probability , weight to the validity of each genotype set as an explanation , rather than merely binary weights as in the combinatorial approaches . a dna or genotype profile is developed from a nucleic acid sample , usually a dna sample . sources of nucleic acid include tissue , blood , semen , vaginal smears , sputum , nail scrapings , or saliva . the dna of interest can be prepared for analysis by amplification and subsequent separation . amplification may be performed by any suitable procedures and by using any suitable apparatus available in the art . for example , enzymes can be used to perform an amplification reaction , such as taq , pfu , klenow , vent , tth , or deep vent . amplification may be performed under modified conditions that include “ hot - start ” conditions to prevent nonspecific priming . “ hot - start ” amplification may be performed with a polymerase that has an antibody or other peptide tightly bound to it . the polymerase does not become available for amplification until a sufficiently high temperature is reached in the reaction . “ hot start ” amplification may also be performed using a physical barrier that separates the primers from the dna template in the amplification reaction until a temperature sufficiently high to break down the barrier has been reached . barriers include wax , which does not melt until the temperature of the reaction exceeds the temperature at which the primers will not anneal nonspecifically to dna . the products of the amplification reaction are detected as different alleles present at a locus or loci . the alleles of at least one locus are amplified and detected after the amplification reaction . if desired , however , the alleles of multiple loci , e . g ., two , three , four , five , six , ten , fifteen , twenty , twenty - five , or thirty , or more different loci may be detected after amplification . sets of loci may include at least two , three , five , ten , fifteen , twenty , thirty , or fifty loci . amplification of all of the alleles may be performed in a single amplification reaction or in a multiplex amplification reaction . alternatively , the sample may be divided into several portions , each of which is amplified with primers that yield product for the alleles present at a single locus . the different alleles at a locus typically are detected because they differ in size . alleles can differ in size due to the presence of repeated dna units within loci . a repeated unit of dna can be , by way of non - limiting example , a dinucleotide , trinucleotide , tetranucleotide , or pentanucleotide repeat . the number of repeated units at a locus also varies . the number of repeated units may be , by way of non - limiting example , at least five , at least ten , at least fifteen , at least twenty , at least twenty - five , or at least fifty units . the effect of these repeated units of dna is the presence of multiple types of alleles that an individual can possess at any given locus that can be detected by size . preferably , alleles that harbor different numbers of str repeat units are detected . more than 8000 strs ( loci ) scattered across the 23 pairs of human chromosomes have been collected in the marshfield medical research foundation in marshfield , wis . preferably , alleles at the 13 core loci used by the fbi combined dna index system ( codis ): csf1po , fga , th01 , tpdx , vwa , d3s1358 , d5s818 , d7s820 , d8s1179 , d13s317 , d16s539 , d18s51 , and d21s11 are detected . it is also contemplated that amplification may be performed to detect an allele by amplifying microsatellite dna repeats , dna flanking alu repeat sequences , or any other known polymorphic region of dna that can be distinguished based on the size of different alleles . the identity of the alleles at one or more of the loci of the reference sample and / or test sample may be determined by short tandem repeat based investigation . whilst the technique is applicable to all loci , the loci for which allele identity is determined may particularly be selected to include one or more of humvwfa31 , humth01 , d21s11 , d18s51 , humfibra , d8s1179 , humamgxa , humamgy , d3s1358 , humvwa , d16s539 , d2s1338 , amelogenin , d8s1179 , d21s11 , d18s51 , d19s433 , humth01 , humfibra / fga . the loci selected may particularly be each of d3s1358 , humvwa , d16s539 , d2s1338 , amelogenin , d8s179 , d21 s11 , d18s51 , d19s433 , humth01 , humfibra / fga . any method that separates amplification products based on size and any method that quantitates the amount of the allele present in the sample can be used to prepare the data required for analysis of genotype profiles in the method . the amplification products may be separated by electrophoresis in a gel or capillary , or mass spectrometry . the amount of each allele present may be determined flourometrically in a flourometer , or via ultraviolet spectrometry . for example , a beckman biomek ® 2000 liquid handling system can be used to detect and quantitate alleles present for a locus in a sample . optical density or optical signal can be used to detect the presence of an allele after gel or capillary electrophoresis . preferably , alleles are detected using an abi prism 310 genetic analyzer , or a hitachi fmbio ii fluorescence imaging system ( 10 ). the abi 310 genetic analyzer identifies alleles present at a locus and provides a data output result . one advantage of this instrument is that , in addition to sizing the detected allele signals , the related software can also display their peak heights and automatically calculate the area under each peak . the hitachi fmbio ii fluorescence imaging system uses gel electrophoresis instead of capillary electrophoresis to separate the alleles of a dna sample . this system requires much more sample and a longer time to complete a separation . in this genetic analyzer , each allele corresponds to a specific band in a gel lane . the band size for each allele is compared with a well - calibrated allelic ladder to identify the corresponding allele . if the amplification products are input into an apparatus that both separates and quantitates alleles for a locus in a sample , four different types of peaks can be obtained from these raw data : true or allele peaks , stutter peaks , artifact peaks , and pull up peaks . exclusion : exclusion from a stain : 1 . a decision ( by the expert ) that a particular reference dna profile does not represent a contributor to the stain ; or , 2 . a situation in which the reference profile is “ excluded ” from the stain at one or more loci . exclusion at a locus : exclusion based on the fact that the pattern of the assumed genotypes at a locus that some allele seen in a particular reference dna profile is not observed in a stain . exclusion probability : the probability that a randomly selected dna profile would be excluded . frequency : rate at which an event occurs . by way of non - limiting example , sample frequency of an allele is the number of occurrences of the allele in a population sample , divided by the sample size ; population frequency of a dna profile is the ( unknown ) number of times that the profile occurs in the population , divided by the population size . a genotype or dna profile is the set of alleles that an individual has at a given locus . a genotype or dna profile may also comprise the sets of alleles that an individual has at more than one locus . by way of non - limiting example , a genotype or dna profile may comprise the set of alleles at each of at least 2 loci , 3 loci , 4 loci , 5 loci , 7 loci , 9 loci , 11 loci , 13 loci , or 20 loci . a genotype profile includes profiles matched to an individual to identify the individual as potentially having contributed to the sample . the genotype profile may be matched to the individual after obtaining a sample from the individual . the genotype profile may also be matched to an individual by comparing it to other genotype profiles in a database . the database may be any public or proprietary database that stores and / or matches genotype profiles . the database may be codis , which may be used to store genotype profiles in a national , state , or regional collection , and which may separate these profiles into disjoint parts , such as a convicted offenders database , a forensic dna database , or a missing persons database . likelihood : conditional probability of an event , where the event is considered as an outcome corresponding to one of several conditions or hypotheses . a non - limiting example of an event is the dna profile evidence from a crime stain . the probability of the event is conditional upon the hypothesis that may vary . if the dna profile is a mixture , a typical prosecution hypothesis may be suspect and victim . this is written as pr ( e | h ), where e is the event , the vertical bar in between the two terms means “ given ”, and h is the hypothesis . likelihood ratio : ratio of two likelihoods , i . e . the ratio of two probabilities of the same event ( e ) under different hypotheses ( h 1 , h 2 ). written as lr =( e | h 1 )/( e | h 2 ). typically h 1 corresponds to the prosecution hypothesis and h 2 corresponds to the defense hypothesis . if h 1 consists of suspect and victim , then the alternative h 2 is unknown and victim . a locus refers to the position occupied by a segment of a specific sequence of base pairs along a gene sequence of dna . genes are differentiated by their specific sequences of base pairs at each locus . an allele refers to the specific gene sequence at a locus . at most two possible alleles can be present at one locus of a chromosome pair for each individual : one contributed by the paternal and the other contributed by the maternal source . if these two alleles are the same , the dna profile is homozygous at that locus . if these two copies are different , the dna profile is heterozygous at the locus . there are multiple alleles that can be contributed by either parent at each locus . minimum peak height ( mph ) is an “ on - the - fly ” variable and will have a value of 150 rfus unless otherwise stated . minimum contributor proportion ( mp ) is an “ on - the - fly ” variable and will have a value of 0 unless otherwise stated . peak height ratio ( phr ) is an “ on - the - fly ” variable and will have a value of 0 . 5 unless otherwise stated . probability : long - term rate of occurrence of an event in a conceptually repeatable experiment . same as expected frequency , the expectation evaluated over cases described by the probability condition ; or , a coherent assignment of a number between zero and one that reflects in a fair and reasonable way our belief that the event is true . proportion ( p ) is the proportion of total rfus of one genotype as compared to the total rfus ( t ). propositions : the hypothesis of the defense or prosecution arguments that are used to formulate the likelihood ratio . a pull - up peak is a false peak reading in a color detection channel at the same place on the x access of a true peak reading at a different color detection channel . the dyes used to label amplified dna fragments fluoresce at different wavelengths . however , there is some overlap in the emission spectra of dyes and , therefore , a blue - labeled dna fragment will also emit a small proportion of green fluorescence . this spectral overlap is mathematically compensated for using software . however , in the case of overamplified samples in a multiplexed process the software can generate a false peak for a color in the spectral overlap . quantitative peak data of ‘ true ’ alleles are determined at a locus . these measurements may be the peak height or peak area of a signal detected by an instrument or procedure designed to quantify the presence of each allele . the peak height , peak area , and any other measurement that is related to the relative masses of each allele present in the original stain or sample are equivalent . quantitative allele peak data will be referred to as “ peak height ,” “ peak area ,” or “ quantitative allele peak data .” each of these terms is interchangeable . restricted combinatorial method : elaboration of the unrestricted method in which allelic intensity ( peak height / area ) information is used to restrict the sets of genotypes that are considered plausible explanations . short tandem repeats ( str ) are dna segments with repeat units of 2 6 bp in length ( 10 ). the repeated unit can be of a longer length that ranges from ten to one hundred base pairs . these are medium - length repeats and may be referred to as a variant number of tandem repeat ( vntr ). repeat units of several hundred to several thousand base pairs may also be present in a locus . these are the long repeat units . stutter : an allelic artifact cause by ‘ slippage ’ of the taq polymerase enzyme . it is always four bases less than the allele that causes the stutter . stutters are always found in allelic positions and can compromise interpretation of minor contributors to mixtures . stutter peaks are peaks generated by the enzyme &# 39 ; s slippage during the amplification process . in most cases , stutter peaks are located on the left side of the associated alleles , and the gene distance between the stutter peak and the associated allele peak is usually less than 4 bp . the height of the stutter peak is usually less than 15 % of the height of the corresponding true allele peak . total rfus ( t ) is the sum of all rfus at the locus of interest . true or allele peaks are peaks that indicate the presence of an allele at a locus . the most important characteristic of an allele peak is that the measured peak area or height is roughly proportional to the mass of the corresponding allele in the dna sample . unrestricted combinatorial method : the simple likelihood ratio method of evaluating mixture evidence described in weir et al . [ 16 ] and clayton and buckleton [ 5 ]. the method assumes a list of all alleles in the mixture , and considers competing hypotheses that various known or unknown profiles are the constituents of the mixture . it uses no information about allelic intensities , hence one set of genotypes whose allele sets are coincident with the mixture is considered to be as valid an explanation of the mixture as any other set . the method disclosed herein removes the analyst bias inherent in known methods by calculating peak height ratios ( phr ) and proportions ( p ) without bias using the same set of calculation rules for every instance . those rules are shown in fig1 . the application of rule 1 is shown on fig2 , wherein a stylized representation of a eletropherogram is shown exhibiting allele peaks corresponding to the a and b allele , with peak heights being measured in rfus . the peak heights , as shown in fig2 for alleles a and b are 1000 and 500 respectively , with the contributing genotypes being aa and ab ( or homozygous and heterozygous ). using rule 3 ( minimum peak heights ), we determine that the peak height difference between alleles a and b is greater than or equal to a predetermined threshold peak ( 150 is the default ). in this case , rule 3 is met ( a − b = 500 ≧ 150 ). under rules 1 and 3 , we are therefore free to assume that the a allele contribution of the ab genotype is equal to the peak height of the b allele , making the ab peak height ratio equal to 1 ( ab phr = 1 ). see fig2 . moving now to rule 2 and fig3 , we see a stylized representation of a electropherogram showing allele peaks corresponding with the a , b and c alleles , peak heights being measures in rfus . the peak heights , as shown in fig3 a for alleles a , b and c are 500 , 1500 and 790 respectively . according to rule 2 , we assume that , for the genotypes ab & amp ; bc combination , the b allele is proportionately shared by the ab and the bc contributions to the dna mixture . taking each allele combination in turn we consider first the amount of contribution of the a and c alleles attributable to genotypes ab & amp ; bc . the proportion of the a allele in the total mixture contribution is a /( a + c )= 500 /( 500 + 790 )= 0 . 39 . the proportion of the c allele in the total mixture contribution is c /( a + c )= 790 /( 500 + 790 )= 0 . 61 . using rule 2 , then , we attribute the level of contribution of the total b allele in the mixture to each genotype ( ab ) and ( bc ) proportionately by their individual ( homozygous allele ) contribution to the mixture as we calculated above . that means that the amount of b allele ( heterozygous ) contribution attributable to the mixture from the ab genotype is calculated as the proportion of a contribution to the total mixture * the total peak height for the b allele in the total mixture , or simply 0 . 39 * 1500 = 585 ( see fig3 b ). similarly , the amount of b allele contribution from the cb genotype is calculated as the proportion of c contribution to the total mixture * the total peak height for the b allele in the total mixture , or simply 0 . 61 * 1500 = 915 ( see fig3 b ). using this calculation and distributing the b allele contributions from the two heterozygous genotypes respectively , we see that the ab peak height ( 500 / 585 = 0 . 85 ) is equal to the and the bc peak height ratio ( 790 / 915 = 0 . 85 ). using the method of proportionate allele sharing as disclosed herein , the ab phr will always equal the bc phr . using the calculations derived from rule 2 we can determine that the proportion of the ab heterozygous genotype contributing to the mixture is the ratio of the total a allele and b allele attributable to the ab genotype ( as calculated above ) and the total rfus in the sample ( for the a , b and c alleles respectively ). this is simply ( 500 + 585 )/ 2 , 790 = 0 . 39 . likewise , we determine the proportion of the bc heterozygous genotype contributing to the mixture is the ratio of the total c allele and b allele attributable to the bc genotype ( as calculated above ) and the total rfus in the sample ( for the a , b and c alleles respectively ). this is simply ( 790 + 915 )/ 2 , 790 = 0 . 61 . moving now to rule 3 and fig4 , we see a stylized representation of a electropherogram showing allele peaks corresponding with the a , and b alleles , peak heights being measures in rfus . the peak heights , as shown in fig4 for alleles a and b are 1000 and 900 respectively . according to rule 3 , minimum peak heights ( mph ) are always maintained and default to 150 rfus . referring now to fig4 a , for the genotype combination ab & amp ; bb , in the case where the difference in peak heights between a and b alleles is less than a predetermined threshold ( with a default of 150 rfus ), we assume that the heterozygous allele b contribution from the bb genotype is equal to the minimum peak height ( mph = 150 ). we also assume that the heterozygous allele b contribution from the ab genotype is equal to the difference between the total b allele rfu level and the minimum peak height . using this assumption , we can calculate the ab phr as equal to ratio of the heterozygous allele b contribution from the ab genotype ( b − mph ) and the total level of a allele in the sample , or simply ( b − mph )/ a =( 900 − 150 )/ 1000 = 0 . 75 . using the assumption from rule 3 , we can also calculate the proportion of contribution of the b allele to the sample mixture from the ab genotype as the ratio of the total a in the mixture plus the rule 3 attributed b allele and the total rfu in the sample , in this case , ( 1000 + 750 )/ 1900 = 0 . 92 . turning now to the application of rule 3 to the instance where a mixture has 3 alleles ( a , b and c ) and to fig5 , we see a stylized representation of a electropherogram showing allele peaks corresponding with the a , b and c alleles , peak heights being measures in rfus . the peak heights , as shown in fig5 for alleles a , b and c are 300 , 400 and 160 respectively . using rule 3 , we assume that the that the heterozygous allele b contribution from the ab genotype is equal to the difference between the total b allele rfu level and the minimum peak height . doing so allows us to calculate the ab phr =( 400 − 150 )/ 300 = 0 . 83 and the ab p =( 300 + 250 )/ t = 0 . 64 . as will be discussed infra , upper and lower boundaries may be calculated in the instance of three - person contributions to preclude combinations that will not allow us to invoke the rule 1 assumption that all peak height ratios equate to 1 . this will be the case where , for example , the ab , and ac genotypes are the major contributors of the b and c alleles and a bc genotype is a minor contributor and vice versa . in such cases , the preferred method allows for upper and lower boundary conditions to be imposed on an individual allele ( in this case , a ) see fig6 . using this method , possible allele combinations will be determined and presented — even if actual ratios and proportions cannot be determined . mph ( mph ) is the user defined minimum peak height used in calculations , the default value is 150 . although not specified in the examples below , the mph is required in every genotype where an allele appears . if there are three contributors with genotypes aa , ab , bc then — 2 * mph total rfus are required for a ( in aa and ab ); 2 * mph total rfus are required for b ( in ab and bc ); and , phr ( phr ) is the user defined minimum peak height ratio used in calculations , the default value is 0 . 5 . for most combinations peak height ratios and contributor proportions can be calculated ; in two instances ( aa and aa ; aa , aa , and aa ) no calculations are performed ; in one instance ( aa , aa and ab ) only a lower boundary is calculated ; in three instances ( aa , ab and bc ; ab , ac and bc ; ab , ac and bd ) both upper and lower boundaries are calculated . when using the 2 or 3 contributor mixture interpretation method , possible combinations are grouped by category of heterozygote and / or homozygote combinations . for abcd alleles : if there are 2 contributors in the mixture , there is one category : ab & amp ; cd ( with possible combinations : ab & amp ; cd , ac & amp ; bd , ad & amp ; bc ); if there are 3 contributors in the mixture , there are 6 categories : aa , bb & amp ; cd ; aa , ab & amp ; cd ; aa , bc & amp ; bd ; ab , ab & amp ; cd ; ab , ac & amp ; ad ; ab , ac & amp ; bd ( with many possible combinations ) for a chart of possible contributor contributions see fig7 and 8 . peak height ratios and proportions , or upper - lower boundaries ( if / when applicable ), are always performed on the entire array of possible combinations within each category . the user may select and set up to six reference samples . when the references are applied , the view of combinations is limited to only those combinations which include the applied references . for a limited selection of the total alleles at a locus ( the user may consider one or more alleles extraneous to the calculation ). for maximum stutter or a user - adjustable 10 to 100 % of the maximum stutter . when evaluating for 2 contributors , ab & amp ; cd calculations are a generic category wherein all combinations within such category ( ab | cd ; ac | bd ; ad | bc ) are always calculated , with only those calculations falling within established parameters being displayed . 6 possibilities for the generic category aa , bb & amp ; cd are calculated ; 12 possibilities for the generic category aa , ab & amp ; cd are calculated ; 12 possibilities for the generic category aa , bc & amp ; bd are calculated ; 6 possibilities for the generic category ab , ab & amp ; cd are calculated ; 4 possibilities for the generic category ab , ac & amp ; ad are calculated ; and , 12 possibilities for the generic category ab , ac & amp ; bd are calculated . aa and aa : no peak height ratio or proportion calculations are performed . aa , aa and aa : no peak height ratio or proportion calculations are performed . ab and cd : a ( 1000 ), b ( 1200 ), c ( 2000 ), d ( 2100 ) ab , ab and cd : a ( 1000 ), b ( 1200 ), c ( 2000 ), d ( 2100 ) ab , cd and ef : a ( 600 ), b ( 700 ), c ( 800 ), d ( 900 ), e ( 1000 ), f ( 1100 ) rule 1 : whenever possible ( while maintaining mph , see rule 3 ), peak height ratios ( phr ) are assumed to equal 1 . if evaluating 2 contributors with genotypes aa & amp ; ab , wherein a rfus - b rfus ≧ mph and assuming a 50 % phr threshold determine how much of the a allele is contributed by the ab genotype : however , if we assume 400 , then 400 / 400 gives a phr = 1 ; therefore , assume 400 rfus are contributed by the ab genotype . see fig9 rule 2 : whenever possible ( while maintaining mph , see rule 3 ), shared alleles are shared proportionately . if evaluating 2 contributors with genotypes ab & amp ; bc , wherein rfus are a ( 1000 ), b ( 1800 ) and c ( 600 ) consider the alleles that will share the c allele : add a b allele and evaluate ab & amp ; bc , ensuring that the b allele is proportionately shared : note that these calculations show proportionate sharing of the b allele ( the percentage of a in the a + c mixture = the percentage of ab in the a + b + c mixture = 0 . 625 ; also the ab phr = the bc phr = 0 . 89 . if evaluating 2 contributors with genotypes aa & amp ; ab , wherein rfus are a ( 1000 ) and b ( 950 ) and the difference between the peak heights is less than the minimum peak height ( mph ), the aa ( homozyote ) peak height is set equal to the mph ( that is , aa = 150 , the default value ). if evaluating 2 contributors with genotypes ab & amp ; bc , wherein rfus are a ( 300 ), b ( 400 ) and c ( 160 ), first determine whether the c allele can be proportionately shared . the calculated portion ( 139 ) is less than the default threshold value for mph ( 150 ), therefore for we set b for the bc equal to the mph ( 150 ) and calculate the remainder contributing portion of the b allele contributed by the ab genotype : the following examples demonstrate how the general calculations used in the forensics community are modified by the three rules disclosed herein . ab , cd and ce : a ( 800 ), b ( 900 ), c ( 1200 ), d ( 800 ), e ( 600 ) determining upper and lower boundaries in situations where ratios and proportions are not calculated in the preferred embodiment the lower boundary for b in ab assumes minimum a in ab ; if a is maximized in aa ( a − mph ), then : if b was & lt ; 500 then rbc would be & lt ; phr or the b in ab would be & lt ; mph if c was & gt ; 700 then the rbc would be & lt ; phr or the b in ab would be & lt ; mph aa , ab and bc : the upper boundary for b : a ( 500 ), b ( 2100 ), c ( 700 ) the upper boundary for b in ab assumes maximum a in ab ; if a is minimized in aa ( mph ), then : could have at most 350 of a ( since a in aa must be at least mph ) could have at most 700 of b ( since a can not be larger than 350 ) for rab = 0 . 5 if b was & gt ; 2100 then rbc would be & lt ; phr or rab could be & lt ; phr if c was & lt ; 700 then the rbc would be & lt ; phr or the rab would be & lt ; phr ab , ac and bc : the lower boundary for a : a ( 400 ), b ( 1200 ), c ( 500 ) the lower boundary for a in ab assumes minimum b in ab ; the lower boundary for a in ac assumes minimum c in ac : if a was & lt ; 1300 then rab would be & lt ; phr or the a in ac would be & lt ; mph ab , ac and bc : the upper boundary for a : a ( 2800 ), b ( 1200 ), c ( 500 ) the upper boundary for a in ab assumes maximum b in ab ; the upper boundary for a in ac assumes maximum c in ac : if a was & gt ; 2800 then rab would be & lt ; phr or the rac would be & lt ; phr if b & lt ; 1200 then rab would be & lt ; phr or the b in bc would be & lt ; mph ab , ac and bd : the lower boundary for b : a ( 500 ), b ( 800 ), c ( 700 ), d ( 1300 ) the lower boundary for b in ab assumes minimum a in ab , and maximum a in ac : must have at least 650 of b for rbd = 0 . 5 ( 150 of b remains ) must have at least 350 of a for rac = 0 . 5 ( 150 of a remains ) if b was & lt ; 800 then the rbd would be & lt ; phr or the b in ab would be & lt ; mph if d was & gt ; 1300 then the rbd would be & lt ; phr or the b in ab would be & lt ; mph if c was & gt ; 700 then the rac would be & lt ; phr or the a in ab would be & lt ; mph if a was & lt ; 500 then the rac would be & lt ; phr or the a in ab would be & lt ; mph ab , ac and bd : the upper boundary for b : a ( 500 ), b ( 2900 ), c ( 700 ), d ( 1300 ) the upper boundary for b in ab assumes maximum a in ab , and minimum a in ac . could have at most 2600 of b for rbd = 0 . 5 ( 300 of b remains ) if b was & gt ; 2900 then the rbd would be & lt ; phr or rab would be & lt ; phr if d was & lt ; 1300 then the rbd would be & lt ; phr or the rab would be & lt ; phr if c was & gt ; 700 then the rac would be & lt ; phr or the a in ab would be & lt ; mph if a was & lt ; 500 then the rac would be & lt ; phr or the a in ab would be & lt ; mph allele a from x unknown contributors ( b , c are from a known contributor ) alleles a , b from x unknown contributors ( c is from a known contributor ) p x ( ab | abc )=( p a + p b + p c ) 2x −( p a + p b ) 2x −( p a + p c ) 2x + p b 2x p x ( abc | abc )=( p a + p b + p c ) 2x −( p a + p b ) 2x −( p b + p c ) 2x −( p a + p b ) 2x + p a 2x + p b 2x + p b 2x p x ( | abc )=( p a + p b + p c ) 2x allele a from x unknown contributors ( b , c , d are from known contributors ) p x ( a | abcd )=( p a + p b + p c + pd ) 2x −( p b + p c + p d ) 2x alleles a , b from x unknown contributors ( c , d are from a known contributor ) p x ( ab | abcd )=( p a + p b + p c + pd ) 2x −( p b + p c + p d ) 2x −( p a + p c + p d ) 2x +( p c + p d ) 2x alleles a , b , c from x unknown contributors ( d is from a known contributor ) ( x & gt ; 1 ) p x ( abc | abcd )=( p a + p b + p c + pd ) 2x −( p b + p c + p d ) 2x −( p a + p c + p d ) 2x −( p a + p b + pd ) 2x +( p c + p d ) 2x +( p b + p d ) 2x +( p a + p d ) 2x − p d 2x p x ( abcd | abcd )=( p a + p b + p c + pd ) 2x −( p b + p c + p d ) 2x −( p a + p c + p d ) 2x −( p a + p b + pd ) 2x −( p a + p b + p c ) 2x +( p c + p d ) 2x +( p b + p d ) 2x +( p b + p c ) 2x +( p a + p d ) 2x +( p a + p c ) 2x +( p a + p b ) 2x − p a 2x − p b 2x − p b 2x − p d 2x p x ( | abcd )=( p a + p b + p c + p d ) 2x the preferred system and method embodiments of this invention are useful for identifying individuals from mixed stains . this has application , for example , in individual identity , where dnas ( e . g ., from people , children , accident victims , crime victims , perpetrators , medical patients , animals , plants , other living things with dna ) may be mixed together into a single mixed sample . then , mixture deconvolution can resolve the mixed data into its component parts . this can be done with the aid of reference individuals , though it is not required . unique identification of individual components of mixed dna samples is useful for finding suspects from dna evidence , and for identifying individuals from dna data in forensic and nonforensic situations . an individual &# 39 ; s genotype can be matched against a database for definitive identification . this database might include evidence , victims , suspects , other individuals in relevant cases , law enforcement personnel , or other individuals ( e . g ., known offenders ) who might be possible candidates for matching the genotype . in one preferred embodiment , the database is a state , national or international dna database of convicted offenders . when there are no ( or only some ) reference individuals , but other information ( such as a database of profiles of candidate component genotypes ) is available , then the invention can similarly derive such genotypes and statistical confidences from the dna mixture data . this is useful in finding suspect individuals who might be on such a database , and has particular application to finding persons ( e . g ., criminals , missing persons ) who might be on such a database . when there is little or no supplementary information , the disclosed method permits computation of probabilities , and evaluation of hypotheses . for example , a likelihood ratio can compare the likelihood of the data under two different models . dna mixtures are currently analyzed by human inspection of qualitative data ( e . g ., electrophoretic bands are present , absent , or something in between ). moreover , they are recorded on databases and reported in court in a similarly qualitative way , using descriptors such as “ major ” or “ minor ” band , and “ the suspect cannot be excluded ” from the mixture . such statements are not optimally compelling in court , and lead to crude database searches generating multiple hits . the system and methods of the preferred embodiment of the invention allow for precise and accurate quantitative analysis of the mixture data to reveal unique identities in many cases . moreover , these mixture analyses can be backed up by statistical certainties that are useful in convincing presentation of evidence . the increased certainty of identification is reflected in the increased likelihood ratios , as well as other probabilities and statistics , as described above . as discussed , with the random person hypothesis of the defense , the current conservative lr analysis weighs heavily in favor of the defense ( national research council , evaluation of forensic dna evidence : update on evaluating dna evidence , 1996 , washington , d . c . : national academy press ), incorporated by reference . the system and analysis disclosed herein help standardize the assumptions made , reduce the potential for examiner error and simplifies the presentation of the evidence , reducing the amount of mathematics that must be explain to the lay juror . the invention includes using quantitative data . this may entail proper analysis or active preservation of the raw str data , including the gel or capillary electrophoresis data files . removing or destroying this highly quantitative information can lead to suboptimal data analysis or lost criminal convictions . the invention enables mathematical estimation of genotypes , together with statistical certainties , that overcome the qualitative limitations of the current art , and can lead to greater certainty in human identification with increased likelihood of conviction in problematic cases . preparing and reviewing reports on mixed dna samples is tedious and time consuming work for the forensic analyst . this dna analysis and reporting expertise is also quite expensive , and represents the single greatest cost in crime laboratory dna analysis . it would be useful to automate this work , including the report generation . this automation has the advantages of higher speed , more rapid turnaround , uniformly high quality , reduced expense , eliminating casework backlogs , alleviating tedium , and objectivity in both analysis and reporting . the system and method of the preferred embodiment are designed for computer - based automation of dna analysis . the results are computed mathematically , and then can be presented automatically as tables and figures via a user interface to the forensic analyst ( see fig1 - 21 ). this analysis and presentation automation provides a mechanism for automated report generation . there is a basic template for reporting dna evidence with which information and analyses that are unique to the case may be merged with information that is generally included . in one preferred embodiment , a template is developed that provides for references to other files and variables . preferable formats include readable documents ( e . g ., word processors , rtf , csv , xlm , xlmt ), hypertext ( e . g ., html ), and other portable document formats ( e . g ., pdf ). a template is a complete document that describes the text and graphics for a standard report , either directly or by reference to variables and files . after the automated mixture analysis , possibly including human review and editing , the computer generates all variables , text , table , figures , diagrams and other presentation materials related to the dna analysis , and preserves them in files ( named according to an agreed upon convention ). the template report document refers to these files , using the agreed upon file naming convention , so that these case - specific materials are included in the appropriate locations in the document . the document preparation program is then run to create a document that includes both the general background and case specific information . this report document , including the case related analysis information ( possibly including tables and figures ), is then preferably output as a bookmarked pdf file . the resulting pdf case report can be electronically stored and transferred , viewed and searched cross platform on local computers or via a network ( lan or wan ), printed , and rapidly provided ( e . g ., via email ) to a crime laboratory or attorney for use as documented evidence . many dna databases permit the inclusion of qualitatively analyzed mixed dna samples . this is particularly true of the “ forensic ” or “ investigative lead ” database components , that contain evidence from unsolved crimes that can be used for matching against dna profiles . when these mixed dna samples are matched against individual or mixed dna queries , many items ( rather than a unique one ) can match . instead of a single dna query uniquely matching a single dna database entry , the dna query can degenerately match a multiplicity of mixed dna database entries . this degeneracy is only compounded when mixed dna queries are made . mixture degeneracy corrupts the database , replacing highly informative unique query matches with large uninformative lists . in these large lists , virtually all the entries are unrelated to the dna query . to prevent this database corruption with mixed dna profiles , it would be useful to clean up the entries prior to their inclusion on the database . when the raw ( or other quantitative ) str data are available , this clean up is readily implemented by the mixture deconvolution invention . for example , consider the common case of a two person mixture containing a known victim and an unknown perpetrator . mixture deconvolution estimates the genotype of the unknown perpetrator , along with a confidence . ( lower confidences may suggest intelligently using degenerate alleles at some loci .) the resolved unknown perpetrator genotypes are then entered into the forensic database , rather than the usual qualitative ( e . g ., major and minor peak ) multiplicity of degenerate alleles . the result is far more uniqueness in subsequent dna query matches , with an associated increase in the informativeness and utility of the matches . when performing dna matches against a dna database , current practice uses mixed dna stains with degenerate alleles . this practice produces degenerate matches , returning lists of candidate matches , rather than a unique match . most ( if not all ) of the entries on this list are typically spurious . the length of these spuriously matching lists grows as the size of the dna database increases . with mixture deconvolution system and method disclosed , the genotype b of an unknown contributor can often be uniquely recovered from the data d and the victim ( s ) a , along with statistical confidence measures . thus , using the resolved mixture b , instead of the qualitative unresolved data d , a unique appropriate database match can be obtained . moreover , the result of this match is highly useful , since it removes the inherent ambiguity of degenerate database matching , and largely eliminates spurious matches . the actual investigative work involved in using the dna evidence to follow leads is very costly as it is so manpower intensive . one reason why this cost is so high is the large number of leads generated by degenerate matches . following one lead is expensive ; following dozens can be prohibitive . and as the sizes of the dna databases increase , the investigative cost of degenerate matches ( from mixed crime stains or mixed database entries ) will increase further . the mixture deconvolution invention overcomes this developing bottleneck . by cleaning up the information prior to its use , the database searching results become more unique and less degenerate . this relative uniqueness translates into reduced investigative work , and greatly reduced costs to society for putting dna technology into practice . in sexual assault cases , differential dna extraction is conducted on semen stains in order to isolate the semen as best as possible . this is done because , a priori , semen stains are considered to be mixed dna samples , and the best possible ( i . e ., unmixed ) evidence is required for finding and convicting the assailant . thus , mixture separation is attempted by laboratory separation processes . the full differential extraction protocols for isolating sperm dna are laborious , time consuming , and expensive . they entail differential cell lysis , and repeatedly performing proteinase k digestions , centrifugations , organic extractions , and incubations ; these steps are followed by purification ( e . g ., using micro concentration ). there are also chelex - based methods . these procedures consume much ( if not most ) of the laboratory effort and time ( often measured in days ) required to for laboratory analysis of the dna sample . this time factor contributes to the backlog and delay in processing rape kits . modified differential dna extraction procedures are also utilized . these procedures eliminate most of the repetitious proteinase k digestions , organic solvent separations , and centrifugations , reducing the total extraction effort from days to hours . however , they do not provide the same degree of separation of the sperm dna template as does the costlier full differential extraction . in fact , highly mixed dna samples will often result . with the mixture deconvolution system and method preferred embodiment , it feasible to expedite the process . the result is the same : the assailant &# 39 ; s sperm cells genotype b is separated from the victim &# 39 ; s epithelial genotype a using the mixed data d . the invention enables crime labs to use faster , simpler and less expensive dna extraction methods , with an order of magnitude difference . the computer performs the refined dna analysis , instead of the lab , resolving the mixture into its component genotypes . to obtain low copy number ( lcn ) data , laboratories will change the pcr protocol , e . g ., increase the cycle number ( say , from 28 to 34 cycles with sgmplus ). experiments are often done in duplicate . the combination of less template and more cycles can lead to increased data artifacts . most prevalent are pcr stutter , allelic dropout , low signal to noise , and mixture contamination . the automated analysis methods described earlier herein readily remove pcr artifacts such as stutter and signal noise . the invention is not dependent on any particular arrangement of the experimental data . in the dna amplification , same dna template is used throughout . for efficiency and consistency of the amplification conditions , a multiplex reaction is preferred . there is no requirement on the specific label or detector used . there is no restriction on the dimensionality of the laboratory system . it can accommodate dimensions of zero ( tubes , wells , dots ), one ( gels , capillaries , mass spectrometry ), two ( gels , arrays , dna chips ), or higher . there is no restriction on the markers or the marker assay used . there are many settings in biology , medicine , and agriculture where mixed dna ( or rna ) samples occur . these samples can be mixed intentionally , or unintentionally , but the problem remains of determining one or more genotype components . in biology , for example , when sequencing dna , it is useful to first sequence the two chromosome sample and then somehow determine the component dna sequences , rather than subclone to first separate and then sequence them . as described herein , the system and method of the preferred embodiment can deconvolve mixed sequences of discrete information , such as dna sequences . in hla typing , for example , the known combinations of sequences permit quantitative information to be resolved using mixture deconvolution . in medicine , cancer cells are a naturally occurring form of dna mixtures . in tumors that exhibit microsatellite instability ( e . g ., from increased str mutation ) or loss of heterozygosity ( e . g ., from chromosomal alterations ), a different typable dna ( the tumor ) is mixed in with the normal tissue . by determining the precise amount of the individual &# 39 ; s normal dna , versus the amount of any other dna ( e . g ., a diverse tumor population ), cancer patients can be diagnosed and monitored using mixture deconvolution . this is done by using the many alleles possibly present at a locus . with diverse tumor tissue subtypes , there may be many alleles present . quantitative data are collected for d , the individual &# 39 ; s known alleles are then used as reference a , and the pattern of the tumor contribution b is determined statistically . another application of the system and method of the present invention is in the deconvolution of biopsies preformed at hospitals and medical facilities . it is often the case that a medical laboratory will perform testing on a number of samples from multiple individuals . the reports that are generated by these medical laboratories may be challenged by the end - user ( i . e . the physician or , more likely , the patient ) as being cross - contaminated with biological material from other sources . using the various methods and systems of the present invention , it is possible to test the underlying biological material used to generate the report to determine whether there has , indeed , been sample convolution . if this proves to be the case , the invention will allow for the deconvolution of the sample to determine which patients have been analyzed . in agriculture , animal materials can be mixed , e . g ., in food , plant or livestock products . the system and method of the preferred embodiment can deconvolve mixed samples into their individual components . in a first preferred embodiment , crime or service laboratories generate their own data from dna samples . the data quantitation and mixture analysis is then done at their site , or , preferably ( from a quality control standpoint ) at a separate data service center ( dsc ). this dsc can be operated by a private for - profit entity , or by a centralized government agency . the case is analyzed , and a report then generated ( in whole or part ) using the software . the report is provided to the originating laboratory . usage fees are applied on a per case basis , with surcharges for additional work . the dsc may provide quality assurance services for provider laboratories to ensure that the data is analyzable by quantitative methods . in a second preferred embodiment , the dsc generates the data , and analyzes it as well . this has the advantage of ensured quality control on the data generation . this can be important when the objective is quantitative data that reflects the output of properly executed data generation . after data analysis , the customer receives the report , and is billed for the case . there are several feasible customers for database work . when entering mixed samples onto a database , it is the database curators and owners ( e . g ., a centralized government related entity ) that is most concerned about the quality of the entered data for future long - term forensic use . this suggests a usage - based contract with said entity for cleaning up the data . a value added by the invention is the capability of finding criminals at a lower cost . when analyzing a mixed dna sample , law enforcement agencies ( e . g ., prosecutors , police , crime labs ) may be interested in identifying genotypes in the mixed sample which are unknown , preferably to match them against a database of possible suspects . in this case , a value added by the invention is the reduced cost , time , and effort of mixture analysis and report generation . there is additional value added in obtaining a higher quality result that can more effectively serve the law enforcement needs of the agency . when matching against a dna database , a single correct match will lead to minimal and successful investigative work by the police or other parties . having a multiplicity of largely incorrect matches creates far greater work , for far less benefit . that is the current art . the invention can ( in many cases ) reduce this work by over an order of magnitude . the value added in this case is the savings in cost and time in the pursuit of justice . when using mixed dna evidence in court , the goal is to obtain a conviction or exoneration , depending on the evidence . the current art produces imprecise , qualitative results that are ill - suited to this purpose . current assessments often vastly understate the true weight of the evidence . the value added in this situation is the capability of the technology to convict the guilty ( and keep them off the street ) and to exonerate the innocent ( and return them to society ). the financial model in this case preferably accounts for the benefit to society of appropriately reduced crime and increased productivity . some embodiments of this invention include a system for resolving a dna mixture comprising : ( a ) means for amplifying a dna mixture , said means producing amplified products ; ( b ) means for detecting the amplified products , said means in communication with the amplified products , and producing signals ; ( c ) means for quantifying the signals that includes a computing device with memory , said means in communication with the signals , and producing dna length and concentration estimates ; ( d ) means for automatically resolving a dna mixture into one or more component genotypes , said means in communication with the estimates ; and ( e ) means for analyzing said estimates and resolutions . fig1 is a flow diagram of a system embodiment of the invention . the advantages of the present invention over the prior are apparent from diagram including , by way of non - limiting example , qa / qc modules for checking ladders , comparing against known references , checking for stutter , checking controls and checking for contamination with cross - references to staff genetic profiles . the novel mixture interpretation method described herein is also incorporated as a module in this system . also included in this system embodiment of the invention are statistical modules for calculating , by way of non - limiting example , single source frequencies , probability of inclusion / exclusion , frequency in mixed samples and likelihood ratios according to the methods disclosed herein . a preferred system embodiment of the invention is shown in fig1 . in this embodiment , the method of this invention is implemented using software running under a secure web server 1 on a protected network 2 that is isolated from a public or private network 3 by a firewall 4 . a remote user located at a database client station 8 may access the implementing software at the web server 1 via the public or private network . the communication may be via the public switched telephone network ( pstn ) preferably using known encryption algorithms for confidential data but is preferably via a private network and encrypted . the firewall 4 allows communications with the secure web server 1 using an encrypted communications protocol such as the hypertext transfer protocol ( http ) over a secure sockets layer ( ssl ). the firewall 4 connects the protected network 2 to the public or private network 3 using either an internet service provider ( isp ), leased , or owned telecommunications equipment / circuits 5 having appropriate bandwidth capability ( although the data may be suitably compressed via known compression algorithms and transmitted over lower bandwidth facilities ). the connection to the firewall 4 and all connections and equipment collocated with the protected network 2 are housed in a secure server facility 6 that provides dna analysis services to a community of clients located at forensic laboratories 7 or other organizations . location 7 , 8 , 9 is shown by way of example only and is no way intended to be limited to forensic laboratory locations . a client 8 located at a forensic laboratory or other organization may use the public or private network 3 to gain access to software services offered by the secure server facility 6 . preferably , the client 8 is connected to a protected network 9 which connects to the public or private network 3 through a firewall 10 , and the firewall 10 , the protected network 9 , and all equipment connected to the protected network 9 , such as the database client 8 , are housed in a secure client facility such as a forensic laboratory 7 ( or other secure facility ). the firewall 10 located at the forensic laboratory 7 connects the protected network 9 to the public or private network 3 using either an isp , leased , or owned telecommunications equipment / circuits 11 having similar bandwidth considerations as described above for equipment / circuits 5 . the client 8 may make requests to analyze data derived from dna mixtures on the secure web server 1 by accessing the secure web server 1 , transmitting dna mixture data to the secure web server , and receiving analysis results . these results may then be interpreted using mixture interpretation guidelines to obtain one or more dna profiles that may be associated with a suspect to a crime . optionally , the database client 8 may access a local laboratory , state , or national dna database 12 to search for matches to the one or more dna profiles formed using the results of the analysis . the dna database 12 may be located in a separate secure facility at the state , local , or national level and is preferentially protected by a firewall 13 . the firewall 13 is connected to the public or private network using either an isp , leased , or owned telecommunications equipment / circuits 14 , and preferentially allows communications with a dna database server 12 using only an encrypted communications protocol such as http over ssl . the firewall 13 and dna database server 12 are connected to a protected network 15 . the connections to the firewall 13 and all connections and equipment collocated with the protected network 15 are housed in a secure server facility 16 that provides dna database services to a community of clients located at forensic laboratories 7 or other organizations . nothing shown in fig1 or described above should be taken to restrict the domain of the invention . for example , the dna database server and the secure service server may be connected through firewalls to two separate and isolated public or private networks , requiring a separate client and protected network located at a forensic laboratory in order to communicate with each server . this is the case at present with the fbi &# 39 ; s national dna index system ( ndis ), which is connected to state and local facilities through the fbi - owned and operated criminal justice information system &# 39 ; s wide area network ( cjis - wan ), and with the current implementation of the secure server . an investigator or analyst transfers results obtained by a client from the secure service server to a client computer of the fbi &# 39 ; s ndis facilities in order to perform a search on the national dna database . the invention is not restricted to operation on protected computers and networks , nor is it restricted to require security of communications using encryption and secure authentication protocols . however , these measures are usually necessitated by the privacy laws of the united states and other countries . in a similar manner , it is not required that the implementing software , database client , and dna database software operate on separate and communicating computers . they may in fact all be installed and operated on a single computer in some applications , or on two computers . there may also be multiple instances of the dna database software running on several computers . the realities of multiple jurisdictions and multiple ownership of and responsibility for controlled access to data that are considered sensitive usually necessitates the use of multiple computers under the control of independent but cooperating agencies . the output of the system embodiment of the invention is shown if fig1 - 20 which was generated using an excel ™ vba application platform ( microsoft , redmond , wash .). however , it is understood that other software vehicle are also appropriate for reproducing the system embodiment of this invention , including , by way of non - limiting example , visual basic ( microsoft , redmond , wash .) and matlab ( mathworks , natick , mass .) implementations . various features of novelty that characterize the invention are pointed out with particularity in the claims annexed to and forming a part of this disclosure . for a better understanding of the invention , its operating advantages and specific objects attained by its uses , reference is made to the accompanying drawings and descriptive in which a preferred embodiment of the invention is illustrated . numerous modifications and variations of the present invention are included in the above - identified specification and are expected to be obvious to one of skill in the art . such modifications and alterations to the compositions and processes of the present invention are believed to be encompassed in the scope of the claims appended hereto . the contents of each of which , and the contents of every other publication , including patent publications such as pct international patent publications , being incorporated herein by this reference .) 1 . mullis , k ., et al ., specific enzymatic amplification of dna in vitro : the polymerase chain reaction . cold spring harb symp quant biol , 1986 . 51 pt 1 : p . 263 - 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