ex vivo method to determine if an alleged father is the biological father of a unborn baby in a preg

专利摘要:
Ex vivo method for determining whether an alleged parent is the biological father of a fetus who is pregnant in a pregnant woman and report the present invention relates to methods for testing noninvasive prenatal paternity are described herein. The method uses genetic measurements made on a pregnant woman's plasma, along with genetic measurements from the alleged father and genetic measurements from the mother to determine whether or not the alleged father is the biological father of the fetus. This is obtained through a computerized method that can compare the genetic profiling of fetal DNA found in maternal plasma with the genetic profiling of the alleged father.
公开号:BR112013016193B1
申请号:R112013016193
申请日:2011-12-22
公开日:2019-10-22
发明作者:Ryan Allison；Gemelos George；Baner Johan；Hill Matthew；Rabinowitz Matthew；Banjevic Milena；Sigurjonsson Styrmir；Demko Zachary
申请人:Natera Inc；
IPC主号:

专利说明:

Invention Patent Descriptive Report for: EX VIVO METHOD FOR DETERMINING IF AN ASSUMED FATHER IS THE BIOLOGICAL FATHER OF A FETUS WHO IS IN PREGNANCY IN A PREGNANT AND REPORT.
RELATED REQUESTS [001] This application claims the benefit of United States Provisional Application Serial No. 61 / 426.208, filed on December 22, 2010 and this application is partly a continuation of United States Utility Application No. Serial 13 / 300,235, filed on November 18, 2011, which claims the benefit of United States Provisional Application No. Serial No. 61/571, 248, filed on June 23, 2011, United States Provisional Application No. 61 / 542.508, filed October 3, 2011 and is a continuation in part of United States Utility Order Serial No. 13 / 110,685, filed on May 18, 2011, which claims the benefit of the Utility Request United States Serial No. 61 / 395,850, filed on May 18, 2010; United States Provisional Application Serial No. 61 / 398,159, filed on June 21, 2010; United States Provisional Application Serial No. 61 / 462,972, filed on February 9, 2011, United States Provisional Application Serial No. 61 / 448,547, filed on March 2, 2011; and United States Provisional Order Serial No. 61 / 516.996, filed on April 12, 2011 and all of these Orders are hereby incorporated by reference with respect to their teachings. FIELD [002] The present invention relates, in general, to methods for testing non-invasive prenatal paternity.
BACKGROUND [003] Uncertain kinship is a significant problem and
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2/116 estimates range between 4% and 10% of children who believe that their biological father is a man who is not their real biological father. In cases where a woman is pregnant, but relevant individuals are not sure who the biological father is, there are several options for determining the correct biological father of the fetus. One method is to wait until birth and carry out the genetic profile of the child and compare the genetic profile of the child's genome with that of the alleged father. However, the mother always wants to know the identity of the biological father of her fetus prenatally. Another method is to perform chorionic villus sampling in the first trimester or amniocentesis in the second trimester and use the removed genetic material to perform the prenatal genetic profile characterization. However, these methods are invasive and present a significant risk of miscarriage.
[004] Recently, it has been discovered that cell-free fetal DNA (cfDNA) and intact fetal cells can enter the maternal bloodstream. Consequently, analysis of this fetal genetic material may allow for Early Non-Invasive Prenatal Genetic Diagnosis (Non-lnvasive Prenatal Genetic Diagnosis - NIPGD or NPD). One of the main challenges in carrying out NIPGD on fetal cells is the task of identifying and extracting fetal cells or nucleic acids from the mother's blood. The concentration of fetal cells in maternal blood depends on the stage of pregnancy and the condition of the fetus, but it is estimated to range from 1-40 fetal cells in every milliliter of maternal blood or less than one fetal cell per 100,000 maternal nucleated cells. Current techniques are able to isolate small amounts of fetal cells from maternal blood, although it is difficult to enrich fetal cells to purity in any amount. The most effective technique in this context involves the use of monoclonal antibodies, but other techniques used to isolate fetal cells
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3/116 include density centrifugation, selective lysis of adult erythrocytes and FACS. A key challenge in carrying out NIPGD on cfDNAfetal is that it is typically mixed with maternal cfDNA and, therefore, the analysis of cfDNA is hampered by the need to consider the maternal genotypic signal. Analysis of fetal DNA has been demonstrated using PCR amplification using primers that are designed to hybridize to sequences that are specific to the paternally inherited genes. These sources of fetal genetic material open the door to non-invasive prenatal diagnostic techniques.
[005] Once the fetal DNA has been isolated, either in a pure state or in a mixture, it can be amplified. There are a number of methods available for amplification of the entire genome (Whole Genome Amplification - WGA): link-mediated PCR (LigationMediated PCR - LM-PCR), PCR with degenerative oligonucleotide primers (Degenerate Oligonucleotide Primer PCR - DOP-PCR) and multiple displacement amplification (Multiple Displacement Amplification - MDA). There are a number of methods available for targeted amplification, including PCR and circularization probes, such as MOLECULAR INVERSION PROBES (MIPs) and PADLOCK probes. There are other methods that can be used, preferably to enrich fetal DNA, such as size separation and hybrid capture probes.
[006] There are numerous difficulties in the use of DNA amplification in these contexts. Amplification of single cell DNA, DNA from a small number of cells or from small amounts of DNA by PCR can fail completely. This is often due to DNA contamination, loss of the cell, its DNA or accessibility of the DNA during the amplification reaction. Other sources of error that can arise in fetal DNA measurement by amplification and microarray analysis include transcription errors
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4/116 introduced by DNA polymerase when a given nucleotide is incorrectly copied during PCR and reading errors due to imperfect hybridization on the array. Another problem is Allele DropOut (ADO), defined as the inability to amplify one of the two alleles in a heterozygous cell.
[007] There are many techniques which provide genotyping data. Some examples include the following. TAQMAN is a unique genotyping technology produced and distributed by LIFE TECHNOLOGY. TAQMAN uses polymerase chain reaction (PCR) to amplify sequences of interest. 500K ARRAYS by AFFYMETRIX and the INFINIUM system by ILLUMINA are genotyping arrangements that detect the presence of specific DNA sequences in a large number of locations simultaneously. HISEQe MISEQ by ILLUMINAe ION TORRENT The LIFE TECHNOLOGY SOLID platform allows the direct sequencing of a large number of individual DNA sequences.
SUMMARY [008] Methods for determining the paternity of an unborn child in a non-invasive manner are described here. According to the aspects illustrated here, in a modality, a method to establish whether a supposed father is the biological father of a fetus who is pregnant a pregnant woman includes obtaining genetic material from the alleged father, obtaining a blood sample from the pregnant woman , carrying out genotypic measurements, in a plurality of polymorphic loci, in the genetic material of the alleged father, obtaining genotypic measurements, in the plurality of polymorphic loci, from the genetic material of the pregnant woman, carrying out genotypic measurements in a mixed DNA sample originating of the blood sample of the pregnant woman, where the mixed DNA sample comprises fetal DNA
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5/116 and maternal DNA, determination, on a computer, of the probability that the alleged father is the biological father of the unborn fetus of the pregnant woman using the genotypic measurements made from the DNA of the alleged father, the genotypic measurements obtained from the pregnant woman and the genotypic measurements made on the mixed DNA sample and establish whether the alleged father is the biological father of the fetus using the determined probability that the alleged father is the biological father of the fetus.
[009] In one embodiment, the polymorphic loci constitute polymorphisms of a single nucleotide. In one embodiment, the mixed DNA sample comprises DNA that was free-floating DNA in a plasma fraction of the pregnant woman's blood sample. In one embodiment, the mixed DNA sample comprises whole maternal blood or a fraction of maternal blood containing nucleated cells. In one embodiment, the fraction of maternal blood containing nucleated cells was enriched for cells of fetal origin.
[0010] In one embodiment, determining whether the alleged father is the biological father includes calculating a test statistic for the alleged father and the fetus, in which the test statistic indicates a degree of genetic similarity between the alleged father and the fetus and on which the test statistic is based on the genotypic measurements made from the DNA of the alleged father, the genotypic measurements made from the mixed DNA sample and the genotypic measurements obtained from the pregnant woman's DNA, calculation of a distribution of a test statistic for a plurality of unrelated individuals to the fetus, where each calculated test statistic indicates a degree of genetic similarity between an unrelated individual to the plurality of individuals unrelated to the fetus and fetus, where the Test statistics are based on genotypic measurements made from unrelated individual DNA, genotypic measurements
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6/116 made from the mixed DNA sample and genotypic measurements obtained from the pregnant woman's DNA, calculating the probability that the test statistic calculated for the alleged father and fetus is part of the distribution of the test statistic calculated for the plurality of unrelated individuals and the fetus and determining the probability that the alleged father is the biological father of the fetus using the probability that the test statistic calculated for the alleged father is part of the distribution of the test statistic calculated for the plurality of unrelated individuals and the fetus. In one embodiment, establishing whether an alleged father is the biological father of the fetus also includes establishing that the alleged father is the biological father of the fetus by rejecting the hypothesis that the alleged father is not related to the fetus if the probability that the supposed father is father is the biological father of the fetus is above a maximum limit or establish that the alleged father is not the biological father of the fetus by not rejecting the hypothesis that the alleged father is not related to the fetus if the probability that the alleged father is the biological father of the fetus is below a minimum limit or does not establish whether an alleged father is the biological father of the fetus if the probability is between the minimum and the maximum limit or if the probability has not been determined with sufficiently high confidence.
[0011] In one embodiment, determining the probability that the alleged father is the biological father of the fetus includes obtaining population frequencies of alleles for each locus of the plurality of polymorphic loci, creating a division of possible fetal DNA fractions in the sample of Mixed DNA ranging from a minimum fetal fraction limit to a maximum fetal fraction limit, calculating the probability that the alleged father is the biological father of the fetus given the genotypic measurements obtained from the mother's DNA, the genotypic measurements made at from the alleged father's DNA, the genotypic measurements made from the mixed DNA sample, for each
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7/116 of the possible fetal fractions in the division, determining the probability that the alleged father is the biological father of the fetus by combining the calculated probabilities that the alleged father is the biological father of the fetus for each of the possible fetal fractions in the division, calculation of a probability that the alleged father is not the biological father of the fetus given the genotypic measurements made from the mother's DNA, the genotypic measurements made from the mixed DNA sample, the population frequencies of alleles obtained for each one of the possible fetal fractions in the division and determining the probability that the alleged father is not the biological father of the fetus by combining the calculated probabilities that the alleged father is not the biological father of the fetus for each of the possible fetal fractions in the division.
[0012] In one embodiment, calculating the probability that the alleged father is the biological father of the fetus and calculating the probability that the alleged father is not the biological father of the fetus can also include calculation, for each of the plurality of polymorphic loci , a probability of sequence data observed at a specific locus using a platform response model, one or a plurality of fractions in the possible fetal fraction division, a plurality of allele proportions for the mother, a plurality of allele proportions for the alleged father and a plurality of allele proportions for the fetus, calculating a probability that the alleged father is the biological father by combining the probability of the sequence data observed at each polymorphic locus over all fetal fractions in the division, on the proportions of the mother's alleles in the set of polymorphic locus, on the supposed proportions of paternal alleles in the set of l polymorphic ocus and the proportions of fetal alleles in the set of polymorphic locus, calculating the probability that the alleged father would not
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8/116 is the biological father by combining the probability of the sequence data observed at each polymorphic locus on all fetal fractions in the division, on the proportions of the mother's alleles in the set of polymorphic loci, on the population frequencies for the set of polymorphic loci and on the proportions of fetal alleles in the set of polymorphic loci, calculating a probability that the alleged father is the biological father based on the probability that the alleged father is the biological father and calculating the probability that the supposed father father is not the biological father based on the likelihood that the alleged father is not the biological father.
[0013] In one embodiment, calculating the probability that the alleged father is the biological father based on the probability that the alleged father is the biological father is performed using a maximum probability estimate or a maximum a posteriori technique. In one embodiment, establishing whether an alleged father is the biological father of a fetus may also include establishing that the alleged father is the biological father if the probability that calculated that the alleged father is the biological father of the fetus is significantly greater than the probability calculated that the alleged father is not the biological father or establish that the alleged father is not the biological father of the fetus if the probability that calculated that the alleged father is the biological father is significantly greater than the calculated probability that the alleged father he is not the biological father. In one embodiment, the polymorphic loci correspond to chromosomes that have a high probability of being disomic.
[0014] In one embodiment, the division of possible fetal DNA fractions contains only a feral fraction and where the fetal fraction is determined by a technique taken from the list consisting of quantitative PCR, digital PCR, targeted PCR, circularization probes, others DNA amplification methods, hybridization probe capture, other preferential enrichment methods,
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9/116 SNP microarrays, DNA microarrays, sequencing, other techniques for measuring polymorphic alleles, other techniques for measuring non-polymorphic alleles, measuring polymorphic alleles that are present in the paternal genome, but are not present in the mother's genome, measurement of non-polymorphic alleles that are present in the paternal genome but are not present in the mother's genome, measurement of alleles that are specific to the Y chromosome, comparison of the measured amount of patently inherited alleles to the measured amount of mathematically inherited alleles, estimates of maximum probability, maximum a posteriori techniques and combinations of the same. In one embodiment, the method according to claim 1, wherein the division of possible fetal fractions contains only a fetal fraction and where the fetal fraction is determined using the method according to claim 26.
[0015] In one embodiment, the genetic material of the alleged father is obtained from tissue selected from the group consisting of: blood, somatic tissue, sperm, hair, oral sample, skin, other forensic samples and combinations thereof. In one embodiment, the trust is computed for the determination established whether the alleged father is the biological father of the fetus. In one embodiment, the fetal DNA fraction in the mixed DNA sample was enriched using a method selected from the group consisting of: size selection, universal PCR-mediated binding, PCR with short extension times, other enrichment methods and combinations thereof.
[0016] In one embodiment, obtaining genotypic measurements from the pregnant woman's genetic material may include making genotypic measurements on a sample of the pregnant woman's genetic material that consists essentially of maternal genetic material. In one embodiment, obtaining genotypic measurements from the material
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10/116 The pregnant woman's genetic data may include inferring which genotypic measurements of the genotypic measurements made on the mixed DNA sample are likely attributable to the pregnant woman's genetic material and use of those genotypic measurements that were inferred to be attributable to the mother's genetic material such as the genotypic measurements obtained. In one embodiment, the method may also include a clinical decision based on an established paternity determination. In one embodiment, the clinical decision is to terminate a pregnancy.
[0017] In one embodiment, genotypic measurements can be made by measuring the genetic material using a technique or technology selected from the group consisting of PADLOCK probes, molecular inversion probes, other circularization probes, genotyping microarrays, SNP genotyping assays, chip-based microarrays, globule-based microarrays, other SNP microarrays, other genotyping methods, Sanger DNA sequencing, pyro-sequencing, high-throughput sequencing, objective sequencing using circularization probes, object sequencing using capture by probe probes hybridization, reversible Dye Terminator sequencing, ligation sequencing, hybridization sequencing, other DNA sequencing methods, other high-throughput genotyping platforms, fluorescent in situ hybridization (Fluorescent In Situ Hybridization - FISH), comparative genomic hybridization (Comparative Genomicic Hybridization - CGH), arrangement CGH and multiples or combinations thereof.
[0018] In one embodiment, genotypic measurements can be made on genetic material that is amplified and / or preferably enriched before being measured using a technique or technology that is selected from the group consisting of:
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Polymerase Chain Reaction PCR, PCR-mediated binding, PCR with degenerative oligonucleotide primer, targeted amplification, mini-PCR, universal PCR amplification, Multiple Displacement Amplification (MDA), allele-specific PCR , allele-specific amplification techniques, linear amplification methods, substrate DNA binding followed by another amplification method, bridge amplification, PADLOCK probes, circularization probes, capture by hybridization probes and combinations thereof.
[0019] In one embodiment, the method may also include generating a report comprising the established paternity of the fetus. In one embodiment, the invention may comprise a report that discloses the established paternity of the fetus generated using a method described here.
[0020] Methods are described here for determining the fraction of DNA from a target individual that is present in a mixture of DNA that contains the target DNA of the individual and also the DNA of at least one other individual. According to aspects illustrated here, in one embodiment, a method for determining a fraction of the DNA of a target individual present in a mixed DNA sample comprising the DNA of the individual and the target DNA of a second individual can include making measurements genotypes of a plurality of polymorphic loci from the mixed DNA sample, obtaining genotypic data in the plurality of polymorphic loci from the second individual and determining, by a computer, the DNA fraction of the target individual present in the mixed sample using the measurements genotypes of the mixed DNA sample, the genotypic data of the second individual and probability estimation techniques.
[0021] In one modality, obtaining genotypic data from the
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12/116 second individual includes making genetic measurements from DNA that essentially consists of the DNA of the second individual. In one embodiment, obtaining genotypic data from the second individual may include inferring which genotypic measurements from the genotypic measurements made on the mixed DNA sample are likely to be attributable to the genetic material of the second individual and use of those genotypic measurements that have been inferred to be attributable to the genetic material of the second individual as the genotypic measurements obtained.
[0022] In one modality, inference of the genotypic data of the related person may also include the use of population frequencies of alleles at the loci. In one embodiment, the DNA fraction determined from a target individual is expressed as a probability of DNA fractions. In one embodiment, the genotypic measurements made on the mixed sample comprise genotypic measurements made by DNA sequencing on the mixed sample. In one embodiment, the DNA of the mixed sample is preferably enriched in the plurality of polymorphic loci before making genotypic measurements of the mixed DNA sample. In one embodiment, the polymorphic loci comprise single nucleotide polymorphisms.
[0023] In one embodiment, fraction determination may also include determining a probability of a plurality of DNA fractions of the target individual present in the mixed DNA sample, determining the fraction by selecting the fraction, from the plurality of fractions, with greater probability. In one embodiment, fraction determination may also include determining a probability of a plurality of DNA fractions from the target individual present in the mixed DNA sample using a maximum probability estimation technique to determine the most likely fraction and
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13/116 fraction determination by selecting the fraction that was determined to be the most likely.
[0024] In one modality, the target individual is a fetus that is pregnant with a pregnant woman and the second individual is the pregnant woman. In one embodiment, the method may also include the use of a platform model that relates measured genotypic data to the polymorphic loci and use of a table that relates maternal genotypes to the child's genotypes. In one embodiment, the determination also uses genotypic measurements at a plurality of polymorphic loci measured on the DNA of the fetus's father. In one embodiment, the method does not make use of genotypic data from the father of the fetus. In one embodiment, the method does not use loci on the Y chromosome. In one embodiment, the invention may comprise a report that discloses an established paternity of the fetus determined using a method described here for determining the fraction of fetal DNA present in maternal plasma. In one embodiment, the invention may comprise a report that discloses a ploidy state of the fetus determined using a method described here for determining the fraction of fetal DNA present in maternal plasma.
BRIEF DESCRIPTION OF THE DRAWINGS [0025] The modalities currently disclosed will be further explained with reference to the attached drawings, in which similar structures are designated by similar numerals throughout the various views. The drawings presented are not necessarily to scale, with emphasis being placed, in general, on the illustration of the principles of the modalities presently described.
[0026] Figure 1 shows the distribution of allele intensities from two parental contexts as measured in maternal plasma.
[0027] Figure 2 shows the distribution of test statistics
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11/14 related to paternity for 200 independent men and the biological father.
[0028] Figure 3 shows two distributions of proportions of intensity for 200 independent men and the biological father. Each graph corresponds to different input channels.
[0029] Figure 4 shows the cumulative distribution frequency curves (Cumulative Distribution Frequency - cdf) for the correlation ratio between the fetal genotypic measurements and the parents' genotypic measurements for three cases.
[0030] Figure 5 shows histograms of the proportion of correlation between fetal genotypic measurements and parental genotypic measurements for three cases.
[0031] Figure 6 shows a histogram of the paternity test statistic for 35 samples when compared to an idealized Gaussian distribution of the test statistic for 800 unrelated men.
[0032] Figure 7 shows an example of a report that describes an exclusion of paternity.
[0033] Figure 8 shows an example of a report that describes an inclusion of paternity.
[0034] Figure 9 shows an example of a report that describes an undetermined result.
[0035] Although the drawings identified above have modalities presently described, other modalities are also considered, as noted in the discussion. This description presents illustrative modalities by way of representation and not limitation. Numerous other modifications and modalities can be conceived by those skilled in the art which fall within the scope and spirit of the principles of the modalities presently described.
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11/15
DETAILED DESCRIPTION [0036] According to the aspects illustrated here, a method is provided to determine whether or not an alleged father is the biological father of a fetus who is pregnant in a pregnant woman. In one embodiment, the method includes obtaining genetic material from the alleged father and obtaining a blood sample from the pregnant woman. In one embodiment, the method may include making genotypic measurements of the alleged father and the pregnant woman and making genotypic measurements on free-floating DNA (ffDNA, that is, cfDNA) found in the pregnant woman's plasma. In one embodiment, the method includes obtaining genotypic data for a set of SNPs from the mother and the alleged father of the fetus; make genotypic measurements for the set of SNPs in a mixed DNA sample comprising the individual's target DNA and also the target individual's mother DNA. In one embodiment, the method may include the use of genotypic measurements to determine, with a computer, the probability that the alleged father is the biological father of the unborn child in the pregnant woman. In one embodiment, the method may include using the genotypic data of the pregnant woman and the alleged father to determine an expected allelic distribution for the genotypic measurements of the fetal / maternal DNA mixture if the alleged father was the biological father of the fetus. In one embodiment, the method may include the use of the pregnant woman's genotypic data and genotypic data from a plurality of individuals known not to be the father to determine an expected allelic distribution for the genotypic measurements of the fetal / maternal DNA mixture if the so-called father is not the biological father of the fetus. In one embodiment, the method may involve calculating the probabilities that the alleged father is the biological father of the fetus given the expected allelic distributions and the actual DNA measurements in the maternal plasma. In one embodiment, the series of steps described in the method results in the transformation of the genetic material of the pregnant woman and the alleged father
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16/116 to produce and determine the correct identity of the biological father of a gestating and prenatal fetus and in a non-invasive way. In one embodiment, determining the probability that the alleged father is the biological father includes assigning or establishing the alleged father as the biological father if the probability that the father is excluded from the allele distribution created using the plurality of unrelated individuals is above of a limit. In one embodiment, determining the probability that the alleged father is the biological father includes attribution of the alleged father that he is not the biological father if the probability that the alleged father is excluded from the allele distribution created using the plurality of individuals without kinship is below the limit. In one embodiment, the determination of paternity is made initially assuming that the alleged father is in fact the child's father; if the alleged parent is incorrect, the child's genotypes will not match these predictions and the initial assumption is considered to be wrong; however, if the child's genotypes match the predictions, then the assumption is considered correct. Thus, the paternity test considers how well the observed ffDNA matches the child's genotypes predicted by the alleged father's genotypes. In one embodiment, an electronic or physical report can be generated indicating the determination of paternity.
[0037] In one embodiment, the determination of paternity is done through genetic measurements of floating DNA (ffDNA) found in the maternal blood and the genotype information of the mother and the alleged father. The general method could be applied to ffDNA measurements using a variety of platforms, such as SNP microarrays, high-throughput targeting or non-targeting sequencing. The methods discussed here address the fact that free floating fetal DNA is found in maternal plasma at low concentrations still unknown and is difficult to
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11/176 detect. The paternity test can include evaluation of ffDNA measurements and the probability that it was generated by the alleged father based on his genotypes. Regardless of the measurement platform, the assay can be based on the genotypes measured at polymorphic locations. In some modalities, the possible alleles at each polymorphic locus can be generalized to A and B and optionally C, D and / or E, etc.
[0038] In one embodiment, this method involves using allele measurement data from a plurality of loci. In one embodiment, the loci are polymorphic. In one embodiment, some or more loci are polymorphic. In one embodiment, the polymorphic loci are polymorphisms of a single nucleotide (Single Nucleotide Polymorphisms - SNPs). In one embodiment, some or most of the polymorphic loci are heterozygous. In one embodiment, it is not necessary to determine which loci are heterozygous before testing. [0039] In one embodiment, a method described here uses selective enrichment techniques that preserve the relative frequencies of alleles that are present in the original DNA sample at each polymorphic locus from a set of polymorphic loci. In some embodiments, the amplification and / or selective enrichment technique may involve PCR techniques, such as mini-PCR or PCR-mediated binding, fragment capture by hybridization or circularization probes, such as Molecular Inversion Probes. In some embodiments, methods for selective amplification or enrichment may involve the use of PCR primers or other probes where, when properly hybridized to the target sequence, the 3 'or 5' end of a nucleotide probe is separated from the polymorphic site of the allele by a small number of nucleotides. In one embodiment, probes in which the hybridization region is designed to hybridize to a polymorphic site are
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11/186 excluded. These modalities are improvements over other methods that involve targeted amplification and / or selective enrichment because they better preserve the frequencies of the sample's original alleles at each polymorphic locus, whether the sample is pure genomics from a single individual or mixture of individuals.
[0040] In one embodiment, a method described here uses highly efficient targeted multiplex PCR to amplify DNA fragments, followed by high throughput sequencing to determine the frequencies of alleles at each target locus. One technique that allows targeted multiplex PCR to be performed in a highly efficient manner involves designing primers that are not susceptible to hybridization to each other. PCR probes can be selected by creating a thermodynamic model of potentially adverse or unwanted interactions between at least 500, at least 1000, at least 5000, at least 10,000, at least 20,000, at least 50,000 or at least 100,000 pairs of primers potentials or between the sample's primers and DNA and then use the template to eliminate designs that are incompatible with the other designs in the group or with the sample DNA. Another technique that allows targeted multiplex PCR to be performed in a highly efficient manner is the use of a partial or total clustering approach to the targeted PCR. Use of one or a combination of these methods allows multiplex formation of at least 300, at least 800, at least 1200, at least 4000 or at least 10,000 primers in a single group with the resulting amplified DNA comprising a majority of the DNA molecules that, when sequenced, it will be mapped to the targeted loci. Use of one or a combination of these methods allows the multiplex formation of a large number of primers in a single
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19/116 group with the resulting amplified DNA comprising more than 50%, more than 80%, more than 90%, more than 95%, more than 98% or more than 99% of DNA molecules that map to the targeted loci.
[0041] In one embodiment, a method described here involves determining whether the distribution of observed allele measurements is indicative of an inclusion or exclusion of paternity using a Maximum Likelihood Estimation (MLE) technique. The use of a maximum probability estimation technique is different from and a significant improvement over methods that use the single hypothesis rejection technique in that the resulting determinations will be made with significantly greater precision. One reason is that single hypothesis rejection techniques do not contain information about the alternative hypothesis. Another reason is that the maximum probability technique allows the determination of the optimal cut-off thresholds for each individual sample. Another reason is that the use of a maximum probability technique allows the calculation of confidence for each determination of paternity. The ability to do a confidence calculation for each determination allows a doctor to know which assignments are accurate and which are most likely to be wrong. In some embodiments, a wide variety of methods can be combined with a maximum probability estimation technique to improve the accuracy of ploidy assignments. In one embodiment, a method described here involves estimating the fraction of fetal DNA in the mixed sample and using the estimate to calculate both the paternity assignment (determination) and the trust in the paternity assignment.
[0042] In one embodiment, the method involves calculating a test statistic that is indicative of the degree of kinship between a first and a second individual given the genotypic measurements in
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20/116 a plurality of polymorphic loci for the first individual and genotypic measurements in a plurality of polymorphic loci for a DNA mixture, where the DNA mixture comprises DNA of the second individual and a related person. In one embodiment, the first individual is an alleged father, the second individual is a gestating fetus and the related person is the mother of the fetus. The test statistic can be calculated for the fetus, the mother and a plurality of individuals known to be unrelated to the fetus, thus generating a distribution of the metric of unrelated individuals. The test statistic can also be calculated for the fetus, the mother and the alleged father. A single hypothesis rejection test can be used to determine whether the test statistic calculated using the alleged father's genotypic data is part of the distribution of test statistics calculated using the genotypic data of unrelated individuals. If the test statistic calculated using genotypic data from the alleged father is found to be part of the distribution of test statistics calculated using the genotypic data of unrelated individuals, then paternity can be excluded, that is, the alleged father can be determined not being related to the fetus. If it is found that the test statistic calculated using genotypic data from the alleged father is not part of the distribution of test statistic calculated using the genotypic data of unrelated individuals, then paternity can be included, that is, the alleged father can be determined to be related to the fetus.
[0043] In one embodiment, the determination of paternity involves determining the probability that the measured genotypic data provide two possible hypotheses: the hypothesis that the alleged father is the biological father of the fetus and the hypothesis that the alleged father is not the father of the fetus. A probability can then be
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21/116 calculated for each of the hypotheses based on the data and paternity can be established based on the probability of each of the two hypotheses. The determination can use genetic measurements made on maternal plasma, measurements made genetic made on the DNA of the alleged father and optionally maternal genotypic data. In one embodiment, maternal genotypic data can be inferred from genotypic measurements performed on maternal plasma. In one embodiment, the probability can be determined using a range division of possible fetal fractions; the range of fetal fractions can be anything from 0.01% to 99.9% and the mesh can have increments ranging from 10% to 1%, from 1% to 0.1% and less than 0.1 %. In one embodiment, the division of possible fetal fractions can be from 2% to 30% and the increments are about 1%. In one embodiment, the mesh may be continuous and the probabilities may be intergrated over the ranges rather than combined. In one embodiment, the probability can be determined using only a fetal fraction, where the fetal fraction can be determined using any appropriate method. For each possible fetal fraction in the mesh, one can calculate the probability of the data indicating two hypotheses. For the hypothesis that the alleged father is the biological father, the genotypes of the alleged father can be used in calculating the probability while, for the hypothesis that the alleged father is not the biological father, population allele frequency data can be used. additionally used in calculating the probability. In one modality, parental contexts and a platform model can be used to calculate the probability of data based on the hypothesis. In one embodiment, the probabilities can be combined for all fetal fractions in the division, all genotypes of the mother and all genotypes of the father. In one embodiment, the parents' genotypes can be probabilistic (for example, in a
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22/116 certain SNP, a parent may have the GT genotype with 99% chances, GG with 0.5% chances and TT with 0.5% chances; in another modality, the parents' genotypes can be based on a value (for example, in a given SNP, one of the parents has the GT genotype). In some modalities, the terms probability and chance can be used interchangeably, as in common language; in other modalities, the two terms may not be used interchangeably and can be read as those versed in the statistical technique would understand.
[0044] In some methods known in the art, the fetal fraction is determined using measurements made at loci that are found exclusively in the paternal genotype, for example, loci that are found exclusively on the Y chromosome or the Rhesus-D gene. Unfortunately, these methods require the fetus to be male (in the case where the loci are found exclusively on the Y chromosome) or a gene or set of genes can be identified before DNA measurements where these genes are present in the paternal genotype and are not present in the maternal genotype. An additional complication is that, in the context of paternity testing, it is not known whether the alleged father is the biological father or not, and therefore, with the exception of the specific Y chromosome loci, it is not possible to determine which loci may be present on the father and not on the mother. Therefore, in the context of paternity testing, it is not really possible to determine the fetal fraction when the fetus is female and, when the fetus is male, the fetal fraction can only be determined using specific Y chromosome loci. one embodiment, a method for determining the fraction of fetal DNA that is present in the DNA mixture comprising maternal and fetal DNA is described here. In one embodiment, the method can determine the fetal fraction of fetal DNA that is present in the
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23/116 DNA mixture comprising maternal and fetal DNA using genotypic measurements of autosomal chromosomes. In one embodiment, the method can determine the fetal fraction of fetal DNA that is present in the DNA mixture comprising maternal and fetal DNA, regardless of the sex of the fetus. In one embodiment, the method can determine the fetal fraction of the fetal DNA that is present in the DNA mixture comprising the maternal and fetal DNA, regardless of what genes the mother and the alleged father may have. The present method does not require the fetus to be male or to identify a locus or loci that are present in the father and not in the mother. The present method does not require that the paternal genotype be known. The present method does not require the mother's genotype to be known, since it can be inferred from measurements made on DNA in maternal plasma, which comprises a mixture of both fetal and maternal DNA.
[0045] In one embodiment, the distribution of polymorphic loci can be modeled using a binomial distribution. In one embodiment, the distribution of polymorphic loci can be modeled using a beta-binomial distribution. Using the betabinomial distribution as a model for the allele distribution, one can model more likely allelic measurements than when using other distributions; this can result in more accurate paternity determinations.
[0046] In one embodiment, a method described here takes into account the tendency for the data to have interference and contain errors when attaching a probability to each measurement. The use of maximum probability techniques to choose the correct hypothesis from the hypothesis set that was made using the measurement data with attached probabilistic estimates makes it more likely that incorrect measurements will be discounted and the correct measurements will be
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24/116 used in calculations that lead to paternity determination. To be more precise, this method systematically reduces the influence of incorrectly measured data in determining paternity. This is an improvement over methods where all data is assumed to be equally correct or methods where peripheral data is arbitrarily excluded from calculations that lead to a determination of paternity. In one mode, individual SN Ps are weighted by the expected measurement variance based on the SNP quality and the observed reading depth; this can result in an increase in the accuracy of the resulting statistic, resulting in an increase in the accuracy of paternity assignments significantly, especially in borderline cases.
[0047] The methods described here are particularly advantageous when used in samples where a small amount of DNA is available or where the percentage of fetal DNA is low. This is due to the correspondingly higher allele abandonment rate that can occur when only a small amount of DNA is available and / or a correspondingly higher fetal allele abandonment rate when the percentage of fetal DNA is accessible in a mixed DNA sample. fetal and maternal. A high allele abandonment rate, meaning that a large percentage of alleles have not been measured for the target individual, results in inaccurate fetal fraction calculations and inaccurate paternity determinations. The methods described here allow an accurate ploidy determination to be made when the percentage of DNA molecules that are fetal in the mixture is less than 40%, less than 30%, less than 20%, less than 10%, less than 8% , less than 6%, less than 4% and even less than 3%.
[0048] In one embodiment, it is possible to determine an individual's paternity based on measurements when the individual's DNA
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11/25 is mixed with the DNA of a related person. In one embodiment, the DNA mixture is the free floating DNA found in maternal plasma, which may include the mother's DNA, with the known genotype and which may be mixed with the fetus' DNA, with an unknown genotype. The fetus' paternity can be determined by looking at the actual measurements and determining the probability of paternity based on the observed data. In some embodiments, a method described here can be used in situations where there is a very small amount of DNA present, such as in forensic situations, where one or a few cells are available (typically less than ten cells, less than twenty cells, less than 40 cells, less than 100 cells or an equivalent amount of DNA). In some embodiments, a method described here can be used in situations where DNA is highly fragmented, such as ffDNA found in plasma. In these modalities, a method described here serves to make paternity assignments from a small amount of DNA that is not contaminated by another DNA, but where the assignment of paternity is very difficult due to the small amount of DNA. The genetic measurements used as part of these methods can be done on any DNA or RNA sample, for example, but without limitations: blood, plasma, body fluids, urine, hair, tears, saliva, tissue, skin, nails, blastomeres, embryos , amniotic fluid, chorionic villus samples, feces, bile, lymph, cervical mucus, semen or other cells or materials comprising nucleic acids. In one embodiment, a method described here can be performed with nucleic acid detection methods, such as sequencing, microarrays, qPCR, digital PCR or other methods used to measure nucleic acids. In some modalities, a method described here involves calculating, by a computer, the allele proportions in the plurality of loci
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26/116 polymorphic from measurements made on the DNA of processed samples. In some embodiments, a method described here involves calculating, by computer, the allele proportions or allelic distributions of a plurality of polymorphic loci from the measurements made on the DNA of the processed samples along with any combination of other enhancements described in the present description. .
[0049] Further discussion of these points can be found elsewhere in this document.
Non-Invasive Prenatal Paternity Testing - NPPT [0050] The process of non-invasive prenatal paternity testing involves a series of steps. Some of the steps may include: (1) obtaining genetic material from the fetus; (2) enrichment of the genetic material of the fetus that may be in a mixed sample ex vivo; (3) amplification of genetic material ex vivo; (4) preferential enrichment of specific loci of genetic material ex vivo; (5) measurement of genetic material ex vivo; and (6) the analysis of genotypic data on a computer and ex vivo. Methods for practicing these steps and six other relevant ones are described here. At least some of the process steps are not directly applied to the body. In one embodiment, the present description refers to methods of treatment and diagnosis applied to tissues and other biological materials isolated and separated from the body. At least some of the steps in the method are performed on a computer.
[0051] Some modalities of the present description allow a doctor to determine the genetic status of a fetus, specifically its biological relationship with another individual, who is gestating in a mother in a non-invasive way, so that the baby's health is not put at risk by collecting genetic material from the fetus and
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27/116 mother is not subjected to an invasive procedure.
[0052] Modern technological advances have resulted in the ability to measure large amounts of genetic information from a genetic sample using methods such as high throughput sequencing and genotyping arrangements. The methods described here allow a physician to take greater advantage of the large amounts of data available and make a more accurate diagnosis of fetal genetic identity. In one embodiment, a computerized method may result in more accurate paternity determinations than methods currently known in the art. Details on a number of modalities are provided below. Different modalities may involve different combinations of the steps mentioned above. Various combinations of the different modalities of the different stages can be used alternately.
[0053] In one embodiment, a blood sample was taken from a pregnant woman and the free floating DNA in the mother's blood plasma, which contains a mixture of maternal DNA and fetal DNA, is isolated and used for determine the fetal ploidy state. In one embodiment, a method described here involves preferential enrichment of these DNA sequences in a mixture of DNA that corresponds to polymorphic alleles in a way that the proportions of alleles and / or allelic distributions remain reasonably consistent when enriched. In one embodiment, the method involves amplifying the isolated DNA using whole genome amplification (Whole Genome Amplification - WGA). In one embodiment, a method described here involves amplification based on targeted PCR, so that a high percentage of the resulting molecules correspond to the targeted loci. In one embodiment, a method described here involves sequencing a mixture of DNA that contains both DNA of maternal origin
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28/116 regarding DNA of fetal origin. In one embodiment, the method involves measuring amplified DNA using a microarray designed to detect nucleic acid sequences, such as an SNP array. In one embodiment, a method described here involves using allele distributions measured to determine the paternity of a fetus that is unborn in a mother. In one embodiment, a method described here involves reporting the determined paternity status to a doctor. In one embodiment, a method described here involves taking a clinical action, for example, performing invasive follow-up testing, such as chorionic villus biopsy or amniocentesis, preparing for the birth of a child or an elective abortion of a fetus.
[0054] This application refers to the North American Utility Application Serial No. 11 / 603,406, filed on November 28, 2006 (North American Publication No. 20070184467); North American Utility Application Serial No. 12 / 076,348, filed on February 17, 2008 (North American Publication No.: 20080243398); PCT Utility Application Serial No. PCT / US09 / 52730, filed on August 4, 2009 (PCT Publication No.: WO / 2010/017214); PCT Utility Application Serial No. PCT / US10 / 050824, filed on September 30, 2010 (PCT Publication No.: WO / 2011/041485), North American Utility Application Serial No. 13 / 110,685, filed on 18 May 2011 and North American Utility Order Serial No. 13 / 300,235, filed on November 18, 2011. Some of the vocabulary used in this presentation may have its background in these references. Some of the terms described here can be better understood in the light of the concepts found in these references. Maternal Blood Screening Understanding Free Floating Fetal DNA [0055] The methods described here can be used to help
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29/116 determine whether a child, fetus or other target individual is genetically related to another individual. In some embodiments, this can be done in cases where the genetic material of the target individual is found in the presence of a quantity of genetic material from another individual. In one embodiment, the method can be used to help determine whether a fetus is genetically related to an alleged father using the free-floating fetal DNA found in maternal blood along with a paternal and optionally genetic sample from the mother. In one embodiment, the fetus may have originated from an egg or an egg donor, so that the fetus is not genetically related to the mother in which the fetus is pregnant. In one embodiment, the method can be applied in cases where the amount of target DNA is in any proportion with the non-target DNA; for example, the target DNA can make up anything between 0.000001 and 99.999999% of the DNA present. In one embodiment, non-target DNA counts! nant may be from a plurality of individuals; it is advantageous where genetic data from some or all relevant non-target individuals are known or where genetic samples from such related individuals are available. In one embodiment, a method described here can be used to determine the genotypic data of the fetus from maternal blood containing fetal DNA. They can also be used if there are several fetuses in the womb of a pregnant woman or where other contaminating DNA may be present in the sample, for example, from others already born siblings.
[0056] This technique can make use of the phenomenon of fetal blood cells that gain access to maternal circulation through the placental villi. Normally, only a very small number of fetal cells enter the maternal circulation in this way (not enough to produce a positive Kleihauer-Betke assay
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30/116 for maternal-fetal hemorrhage). Fetal cells can be classified and analyzed using a variety of techniques to search for specific DNA sequences, but without the risks inherent in invasive procedures. This technique can also make use of the free floating feral DNA phenomenon that gains access to maternal circulation by releasing DNA after apoptosis of placental tissue, where the placental tissue in question contains DNA from the same genotype as the fetus. The free-floating DNA found in maternal plasma has been shown to contain fetal DNA in proportions as high as 30-40% fetal DNA.
[0057] In one mode, blood can be collected from a pregnant woman. Research has shown that maternal blood may contain a small amount of free-floating fetal DNA, in addition to free-floating DNA of maternal origin. In addition, there may also be nucleated fetal blood cells comprising DNA of fetal origin, in addition to several blood cells of maternal origin which, typically, do not contain nuclear DNA. There are many methods known in the art to isolate fetal DNA or create fractions enriched in fetal DNA. For example, it has been shown that chromatography creates certain fractions that are rich in fetal DNA.
[0058] Once the sample of maternal blood, plasma or other fluid, taken in a relatively non-invasive way and that contains a quantity of fetal DNA, either cellular or free-floating, is enriched in its proportion to the maternal DNA or its original proportion, is at hand, genotyping of the DNA found in said sample can be performed. In some modalities, blood can be collected with a needle to draw blood from a vein, for example, the basilic vein. The method described here can be used to determine fetal genotypic data. For example, it can be used to determine the state of ploidy in one or more
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11/116 chromosomes, can be used to determine the identity of one or a set of SNPs, including insertions, deletions and translocations. It can be used to determine one or more haplotypes, including the parent of origin of one or more genotypic characteristics. It can also be used to determine the degree of kinship between the fetus and another individual.
[0059] Note that this method will work with any nucleic acids that can be used for any genotyping and / or sequencing methods, such as the ILLUMINA INFINIUM ARRAY, AFFYMETRIX GENECHIP, ILLUMINA GENOME ANALYZER or SOLID SYSTEM platform from LIFE TECHNOLGIES, together with genotypic data measured by them. This includes free-floating DNA extracted from the plasma or amplifications (eg, whole genome amplification, PCR); Genomic DNA from other types of cells (for example, human whole blood lymphocytes) or amplifications of them. For DNA preparation, any extraction or purification method that generates genomic DNA suitable for one of these platforms will work well. This method could work equally well with RN A samples. In one embodiment, sample storage can be done in a way that minimizes degradation (for example, below zero, at about -20 ° C or at a lower temperature).
PARENTAL SUPPORT® [0060] Some modalities can be used in combination with the PARENTAL SUPPORT® (PS) method, modalities of which are described in North American Order No. 11 / 603.406 (North American Publication No.: 20070184467), North American Order No. 12 / 076,348 (North American Publication No.: 20080243398), North American Application No. 13 / 110,685, PCT Application PCT / US09 / 52730 (PCT Publication No.: WO / 2010/017214), PCT Application No. PCT / US10 / 050824
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32/116 (PCT Publication No.: WO / 2011/041485), PCT Application No. PCT / US2011 / 037018 (PCT Publication No.: WO / 2011/146632) and PCT Application No. PCT / US2011 / 61506, which are hereby incorporated by reference in their entirety. PARENTAL SUPPORT® is a computerized approach that can be used to analyze genetic data. In some embodiments, the methods described here can be considered as part of the PARENTAL SUPPORT ™ method. In some embodiments, the PARENTAL SUPPORT ™ method is a set of methods that can be used to determine genetic data from an individual target, with high precision, from one or a small number of cells of that individual or a mixture of DNA consisting of DNA of the target individual and DNA of one or a plurality of other individuals, specifically to determine disease-related alleles, other alleles of interest, the ploidy state of one or a plurality of target chromosomes in the individual and / or the extent of relationship in another individual with the target individual. PARENTAL SUPPORT ® can refer to any of these methods. PARENTAL SUPPORT ® is an example of a computerized method.
[0061] The PARENTAL SUPPORT ® method makes use of genetic data from known parents, that is, haplotypic and / or diploid genetic data from the mother and / or father, together with knowledge of the mechanism of meiosis and imperfect measurement of the target DNA and possibly one or more related individuals, along with population-based cutoff frequencies in order to reconstruct, in silico, the genotype in a plurality of alleles and / or the paternity status of an embryo or any (any) target cell (s) and target DNA in locating major loci with a high degree of confidence. The PARENTAL SUPPORT® method makes use of data known to the genetic parents, that is, haplotype data and / or
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33/116 mother and / or father diploid genetics, along with knowledge of the mechanism of meiosis and imperfect measurement of target DNA, to create hypotheses about what genetic data can be expected for different situations to calculate the probability of each of the situations provided in the observed genetic data, thus determining which situation is most likely. In some embodiments, the situation is question may include whether the target individual has inherited a haplotype linked to the disease of interest, whether the target individual has inherited a haplotype linked to the phenotype of interest, whether the target individual has one or more aneuploid chromosomes and / or the target individual is related to an individual of interest and what the degree of relatedness may be. The PARENTAL SUPPORT® method allows the cleaning of interference genetic data. PARENTAL SUPPORT® may be particularly relevant where only a small fraction of the available genetic material is from the target individual (for example, NPD or NPPT) and where direct measurements of the genotypes inherently interfere due to the contaminating DNA signal from another individual . The PARENTAL SUPPORT® method is capable of reconstructing diploid allele sequences ordered in high precision over the embryo, together with the number of copies of chromosomal segments, even though conventional unordered diploid measurements can be characterized by high rates of abandonment of alleles, evasions , variable amplification trends and other errors. The method can use either an underlying genetic model or an underlying measurement error model. The genetic model can determine the probabilities of alleles in each SNP and cross-probabilities between SNPs. Allele probabilities can be modeled in each SNP based on data obtained from parents and cross the probability model between SNPs based on data obtained from the HapMap database, developed by
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11/36
Intemational HapMap Project. Given the appropriate underlying genetic model and measurement error model, maximum a posteriori estimation (MAP) can be used, with modifications for computational efficiency, to estimate the correct ordered allele values for each SNP in the embryo.
Definitions [0062] Polymorphism of a Single Nucleotide (Single Nucleotide Polymorphism - SNP) refers to a nucleotide that can be different between the genomes of two members of the same species. The use of the term does not imply any limit to the frequency with which each variant occurs.
[0063] Sequence refers to a DNA sequence or a genetic sequence. It can refer to the primary physical structure of the DNA molecule or strand in an individual. It can refer to the nucleotide sequence found in that DNA molecule or to the complementary strand of the DNA molecule. It can refer to the information contained in the DNA molecule as its in silico representation.
[0064] Locus refers to a specific region of interest on an individual's DNA, which can refer to a SNP, the location of a possible insertion or deletion or the location of some other relevant genetic variation. SN Ps related to the disease can also refer to loci linked to the disease.
[0065] Polymorphic allele, also polymorphic locus, refers to an allele or locus where the genotype varies between individuals of a given species. Some examples of polymorphic alleles include single nucleotide polymorphisms, short random repetitions, deletions, duplications and inversions.
[0066] Polymorphic Site refers to specific nucleotides found in a polymorphic region that varies between individuals.
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35/116 [0067] Allele refers to genes that occupy a particular locus.
[0068] Genetic Data, also Genotypic Data, refers to data that describe aspects of the genome of one or more individuals. They can refer to one or a set of loci, partial or whole sequences, partial or whole chromosomes or the entire genome. They can refer to the identity of one or a plurality of nucleotides; they can refer to a set of sequential nucleotides or nucleotides from different locations in the genome or a combination of them. Genotypic data is typically in silico, however, it is also possible to consider physical nucleotides of a sequence as chemically encoded genetic data. Genotypic data can be said to be on, from, on, from or on the individual (s). Genotypic data can refer to measurements resulting from a genotyping platform where these measurements are made on genetic material.
[0069] Genetic Material, also a Genetic Sample, refers to the physical matter, such as tissue or blood, of one or more individuals comprising DNA or RNA.
[0070] Trust refers to the statistical probability that the assigned SNP, allele, set of alleles, ploidy assignment or paternity assignment is correct.
[0071] Aneuploidy refers to the state where the wrong number of chromosomes is present in a cell. In the case of a somatic human cell, it can refer to the case where a cell does not contain 22 pairs of autosomal chromosomes and one pair of sex chromosomes. In the case of a human gamete, it can refer to the case where a cell does not contain one of each of the 23 chromosomes. In the case of a single type of chromosome, it can
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36/116 refer to the case where more or less than two counterparts, but not identical chromosomal copies, are present or where there are two copies of the chromosome present that originate from the same parent. [0072] Chromosome can refer to a single chromosomal copy, that is, a single DNA molecule, of which there are 46 in a normal somatic cell; an example is chromosome 18 of maternal origin. Chromosome can also refer to a type of chromosome, of which there are 23 in a normal human somatic cell; an example is chromosome 18.
[0073] Monosomy refers to the state where a cell contains only one of a kind of chromosome.
[0074] Disomy refers to the state where a cell contains two of a chromosome type.
[0075] Uniparental disomy refers to the state where a cell contains two of a type of chromosome and where both chromosomes originate from one parent.
[0076] Trisomy refers to the state where a cell contains three of a chromosome type.
[0077] The State of Genetic Material or simply Genetic State can refer to the identity of a set of SNPs on DNA, to the haplotypes eliminated in the genetic material or to the DNA sequence, including deletions, insertions, repetitions and mutations. It can also refer to the ploidy state of one or more chromosomes, chromosomal segments or set of chromosomal segments.
[0078] Establishment of Paternity or Determination of Paternity refers to the establishment or determination whether or not an alleged father is the biological father of an unborn child or determining or establishing the likelihood that an alleged father is the biological father of the fetus .
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37/116 [0079] Determination of Paternity refers to the determination of whether the alleged father is the biological father of the fetus or not. A paternity determination is the result of establishing, assigning or determining paternity.
[0080] Paternity refers to the identity of an individual's biological father.
[0081] Inclusion of Paternity refers to the establishment that an alleged father is the biological father of a fetus.
[0082] Exclusion of Paternity refers to the establishment that an alleged father is not the biological father of a fetus.
[0083] Supposed Father refers to a man whose paternal relationship with a fetus is in question.
[0084] Biological Father of an individual refers to the man whose genetic material was inherited by the individual.
[0085] Allelic Ratio refers to the ratio between the quantity of each allele in a polymorphic locus that is present in a sample or in an individual. When the sample is measured by sequencing, the allelic proportion can refer to the proportion of sequence read that maps to each allele in the locus. When the sample is measured using an intensity-based measurement method, the allele ratio can refer to the proportion between the quantities of each allele present in this locus, as estimated by the measurement method.
[0086] Allele Distribution or Count Distribution Allele refers to the relative quantity of each allele that is present for each locus in a set of loci. An allelic distribution can refer to an individual, a sample or a set of measurements made on a sample. In the context of sequencing, allelic distribution refers to the number or probable number of readings that map to a particular allele for each allele in a set
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38/116 of polymorphic loci. Allele measurements can be treated probabilistically, that is, the probability that a given allele is present to provide a sequence reading is a fraction between 0 and 1 or can be treated in a binary way, that is, any given reading is considered to be exactly zero or a copy of a particular allele.
[0087] Allelic Tendency refers to the degree to which the measured proportion of alleles in a heterozygous locus is different from the proportion that was present in the original DNA sample. The degree of allelic tendency at a particular locus is equal to the allelic proportion observed at this locus, as measured, divided by the proportion of alleles in the original DNA sample at this locus. Allelic trend can be defined as being greater than one so that if the calculation of the degree of allelic trend resumes to an x value that is less than 1, then the degree of allelic trend can be reset to 1 / x. Allelic tendency may be due to amplification tendency, purification tendency or some other phenomenon that affects different alleles differently.
[0088] Primer, also PCR probe refers to a single DNA molecule (a DNA oligomer) or a set of DNA molecules (DNA oligomers) where the DNA molecules are identical or nearly identical and where the primer contains a region that is designed to hybridize to a target polymorphic locus and can contain an initiation sequence designed to allow PCR amplification. A primer can also contain a molecular barcode. A primer can contain a random region that is different for each individual molecule.
[0089] Hybrid Capture Probe refers to any nucleic acid sequence, possibly modified, that is generated by various methods, such as PCR or direct synthesis, and is intended to be
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39/116 complement a strand of a specific target DNA sequence in a sample. The exogenous hybrid capture probes can be added to a previously prepared sample and hybridized through a denaturation-reseal process to form pairs of exogenous / endogenous fragments. These pairs can then be physically separated from the sample by various means. [0090] Sequence Reading refers to data representing a sequence of nucleotide bases that were measured using a clonal sequencing method. Clonal sequencing can produce sequence data that represents a single clone or clones or clusters of the original DNA molecule. A sequence reading may also have associated the quality score at each position in the sequence that indicates the probability that a nucleotide has been assigned correctly.
[0091] Mapping a Sequence Reading is the process of determining the location of a source sequence reading in the genomic sequence of a particular organism. The origin location of the sequence readings is based on the similarity of the reading nucleotide sequence and genomic sequence.
[0092] Homozygous refers to having similar alleles at the corresponding chromosomal loci.
[0093] Heterozygote refers to having different alleles at the corresponding chromosomal loci.
[0094] Heterozygosity Rate refers to the rate of individuals in the population having heterozygous alleles at a given locus. The rate of heterozygosity can also refer to the expected or measured proportion between alleles at a given locus in an individual or in a DNA sample.
[0095] Haplotype refers to a combination of alleles at multiple loci that are usually inherited together over the same
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40/116 chromosome. The haplotype can refer to only two loci or an entire chromosome, depending on the number of recombination events that have occurred between a given set of loci. Haplotype can also refer to a set of polymorphisms of a single nucleotide (Single Nucleotide Polymorphisms - SNPs) on a single chromatid that are statistically associated.
[0096] Haplotypic data, also Deleted Data or Ordered Genetic Data, refers to data from a single chromosome in a diploid or polyploid genome, that is, either the secreted maternal or paternal copy of a chromosome in a diploid genome.
[0097] Elimination refers to the act of determining an individual's haplotype genetic data based on disordered diploid (or polyploid) genetic data. It can refer to the act of determining which of the two genes in an allele, for a set of alleles found on a chromosome, are associated with each of the two homologous chromosomes in an individual.
[0098] Deleted Data refers to genetic data where one or more haplotypes have been determined.
[0099] Fetal refers to that of the fetus or the region of the placenta that is genetically similar to the fetus. In a pregnant woman, a part of the placenta is genetically similar to the fetus and the free-floating fetal DNA found in maternal blood may have originated from the part of the placenta with a genotype that matches the fetus.
[00100] DNA of Fetal Origin refers to DNA that was originally part of a cell whose genotype was essentially equivalent to that of the fetus. It should be noted that the genetic information on half of a fetus' chromosomes is inherited from the mother of the fetus; in some modalities, the DNA of these maternally inherited chromosomes that originate from a fetal cell is
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41/116 considered to be of fetal origin and not of maternal origin. [00101] DNA of Maternal Origin refers to DNA that was originally part of a cell whose genotype was essentially equivalent to that of the mother.
[00102] Child can refer to an embryo, a blastomer or a fetus. It should be noted that, in the modalities presently described, the concepts described apply equally to individuals who are of a newborn child, a fetus, an embryo or a set of cells resulting from it. The use of the term child may simply be meant to denote that the individual referred to as the child has the genetic ancestry of the parents.
[00103] Parental refers to the genetic mother or father of an individual. An individual generally has two parents, a mother and a father, although this may not necessarily be the case, as in genetic or chromosomal chimerism.
[00104] Mother can refer to the biological mother of an individual and / or it can refer to the woman who is bringing the individual that she gestates.
[00105] Parental Context refers to the genetic status of a given SNP on each of the two chromosomes relevant to one or both parents of the target.
[00106] Plasma Materno refers to the plasma portion of the blood of a woman who is pregnant.
[00107] Clinical Decision refers to any decision to take or not to take an action that has a result that affects the health or survival of an individual. In the context of prenatal paternity testing, a clinical decision may refer to a decision to abort or not to abort a fetus. The clinical decision may also refer to the decision to perform further tests or to take steps to prepare for the birth of a child.
[00108] Diagnosis box refers to one or a combination
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42/116 of machines designed to perform one or a plurality of aspects of the methods described here. In one embodiment, the diagnostic box can be placed at a point of care for the patient. In one embodiment, the diagnostic box can perform targeted amplification, followed by sequencing. In one embodiment, the diagnostic box can work alone or with the help of a technician.
[00109] Computerized Method or Computerized Approach refers to a method that relies heavily on statistics to make sense of a large amount of data. In the context of prenatal diagnosis, it refers to a method designed to determine the state of ploidy in one or more chromosomes or the allelic state in one or more alleles by statistically inferring the most likely state, rather than measuring the state directly based on a large amount of genetic data, for example, from a molecular arrangement or sequencing. In an embodiment of the present description, the computerized technique can be one described in this patent. In one embodiment of the present description, it can be PARENTAL SUPPORT®.
[00110] Preferential enrichment of DNA that corresponds to one or a plurality of loci or preferential enrichment of DNA in one or a plurality of loci refers to any method that results in the percentage of DNA molecules in a post-DNA mixture enrichment that corresponds to the loci that is greater than the percentage of DNA molecules in the mixture of pre-enrichment DNA that correspond to the loci. The method may involve selective amplification of DNA molecules that correspond to the loci. The method may involve removing DNA molecules that do not correspond to the loci.
[00111] Amplification refers to a method that increases the number
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43/116 copies of a DNA molecule.
[00112] Selective Amplification can refer to a method that increases the number of copies of a given DNA molecule or DNA molecules that correspond to a particular DNA region. It can also refer to a method that increases the number of copies of a given target DNA molecule or target DNA region increases more than non-target DNA molecules or regions. Selective amplification can be a preferred enrichment method.
[00113] Universal Production Sequence refers to a DNA sequence that can be added to a population of target DNA molecules, for example, by ligation, by PCR or PCR-mediated ligation. Once added to the target molecule population, primers specific to universal production sequences can be used to amplify the target population using a single pair of amplification primers. Universal production sequences are usually not related to the target sequences.
[00114] Universal Adapters or Link Adapters or Library Tags are DNA molecules containing a universal production sequence that can be covalently linked to the 5 'and 3' ends of a population of double stranded DNA molecules. The addition of the adapters allows universal production sequences at the 5 'and 3' ends of the target population from which PCR amplification can occur, amplifying all molecules from the target population using a single pair of amplification primers.
[00115] Objectification refers to a method used to selectively amplify or otherwise enrich preferably those DNA molecules that correspond to a set of loci in a
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44/116 mixing of DNA.
[00116] H / poster refers to the possibility that the alleged father is the biological father of the fetus or that the alleged father is not the biological father of the fetus.
[00117] Determination, establishment and calculation can be used alternately.
Parental Contexts [00118] The parental context refers to the genetic status of a given allele on each of the two chromosomes relevant to one or both of the target's parents. It should be noted that, in one mode, the parental context does not refer to the target's allelic state; rather, it refers to the parents' allelic state. The parental context for a given SNP can consist of four base pairs, two paternal and two parent; they can be the same or different from each other. It is usually written as mim2 | fif2, where mi and nr) 2 are the genetic state of a given SNP on the two chromosomes matemos and fi ef ₂ are the genetic state of a given SNP on the two paternal chromosomes. In some modalities, the parental context can be written as fif2 | mim2. Note that subscripts 1 and 2 refer to the genotype, in a given allele, of the first and second chromosomes; note also that the choice of which chromosome is marked as 1 and which is marked as 2 may be arbitrary.
[00119] Note that, in the present description, A and B are often used to generically represent base pair identities; A or B could also represent C (cytosine), G (guanine), A (adenine) or T (thymine). For example, if, in a given SNP-based allele, the mother's genotype was T in this SNP on a chromosome and G in this SNP on the homologous chromosome and the paternal genotype in this allele is G in this SNP over both
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45/116 homologous chromosomes, it can be said that the target individual's allele has the parental context of AB | BB; but it could also be said that the allele has the AB | AA parental context. Note that, in theory, any of the four possible nucleotides could occur in a given allele, so it is possible, for example, that the mother has an AT genotype and the father has a GC genotype in a given allele. However, empirical data indicates that, in most cases, only two of the four possible base pairs are observed in a given allele. It is possible, for example, when using single random repetitions, to have more than two parental contexts, more than four and even more than ten. In the present description, the discussion assumes that only two possible base pairs will be observed in a given allele, although the modalities described here can be modified to take into account cases where such an assumption is not supported.
[00120] A parental context can refer to a set or subset of target SNPs that have the same parental context. For example, if you were to measure 1000 alleles on a given chromosome on an individual target, then the AA | BB context could refer to the set of all alleles in the 1,000 allele group where the target mother's genotype was homozygous and the genotype of the target's father is homozygous, but where the maternal genotype and the paternal genotype are different in this locus. If parental data is not deleted and therefore AB = BA, then there are nine possible parental contexts: AA | AA, AA | AB, AA | BB, AB | AA, AB | AB, AB | BB, BB | AA , BB | AB and BB | BB. If paternal data is deleted and therefore AB Ψ BA, then there are sixteen different possible parental contexts: AA | AA, AA | AB, AA | BA, AA | BB, AB | AA, AB | AB, AB | BA , AB | BB, BA | AA, BA | AB, BA | BA, BA | BB, BB | AA, BB | AB, BB | BA and BBjBB. Each SNP allele on a chromosome, excluding some
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SNPs on sex chromosomes, have one of these parental contexts. The set of SNPs in which the parent context for a parent is heterozygous can be referred to as the heterozygous context.
Different implementations of the methods Currently disclosed [00121] Method are disclosed here to determine the paternity of a target person. The individual may be a target of blastomeres, an embryo or a fetus. In some embodiments of the present description, a method for determining an individual's paternity may include any of the steps described in this document and combinations thereof:
[00122] In some embodiments the source of genetic material to be used in determining the paternity of the fetus may be fetal cells, such as fetal nucleated red blood cells, isolated from maternal blood. The method may involve obtaining a blood sample from the pregnant woman. In some embodiments of the present description, the genetic material to be used in determining the paternity of the fetus can release the floating DNA from maternal plasma, where the free floating DNA can be made up of a mixture of fetal and maternal DNA.
[00123] In some embodiments, the source of the genetic material of the fetus may be fetal cells, such as fetal nucleated red blood cells, isolated from maternal blood. The method may involve taking a blood sample from the pregnant woman. The method may include isolating a fetal red blood cell using visual techniques, based on the idea that a given color combination is exclusively associated with nucleated red blood cells and a similar color combination is not associated with any other cell present in the maternal blood. THE
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47/116 color combination associated with nucleated red blood cells may include the red color of hemoglobin around the nucleus, which the color can be made more distinct by staining and the color of the nuclear material, which can be stained, for example, blue. By isolating the maternal blood cells and spreading them over a slide and then identifying the points at which both red (from hemoglobin) and blue (from nuclear material) are seen, one may be able to identify the location of nucleated red blood cells. You can then extract the nucleated red blood cells, using a micromanipulator, use genotyping and / or sequencing techniques to measure aspects of the genotype of the genetic material in the cells.
[00124] In one embodiment, the nucleated red blood cell can be stained with a die that only fluoresces in the presence of fetal hemoglobin, non-maternal hemoglobin and thus eliminating the ambiguity between whether a nucleated red blood cell is derived from the mother or the fetus. Some embodiments of the present description may involve staining or otherwise marking the nuclear material. Some embodiments of the present description may specifically involve labeling of fetal nuclear material, using antibodies specific to fetal cells.
[00125] There are many other ways to isolate fetal cells from maternal blood or fetal DNA from maternal blood or to enrich samples of fetal genetic material in the presence of maternal genetic material. Some of these methods are listed here, but this is not intended to be an exhaustive list. Some appropriate techniques are listed here for convenience: using fluorescent or unlabeled antibodies, size exclusion chromatography, magnetically or unlabeled affinity tags, epigenetic differences, such as differential methylation between maternal cells and
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48/116 fetuses of specific alleles, density gradient centrifugation followed by CD45 / 14 exhaustion and positive CD71 selection from CD45 / 14 negative cells, individual or double Percoll gradients with different osmolalities or specific galactose lectin method. [00126] In some modalities, the genetic sample can be prepared, isolated and / or purified. In some embodiments, the sample can be centrifuged to separate several layers. In some modalities of DNA preparation it may involve amplification, separation, purification by chromatography, electrophoresis purification, filtration, liquid-liquid separation, isolation, precipitation, preferential enrichment, preferential amplification, target amplification or any one of a number of other techniques or known in the art or described herein.
[00127] In some embodiments, the method of the present description may involve amplification of DNA. DNA amplification, a process that transforms a small amount of genetic material into a larger amount of genetic material that comprises a similar set of genetic data, can be done by a variety of methods, including, but not limited to, chain reaction polymerase (PCR). One method of DNA amplification is the amplification of the entire genome (WGA). There are a number of methods available for WGA: PCR-mediated binding (LM-PCR), PCR primer degenerate oligonucleotide (DOP-PCR), displacement and multiple amplification (MDA). In LM-PCR, short DNA sequences called adapters are attached to smooth ends of DNA. These adapters contain universal amplification sequences, which are used to amplify DNA by PCR. In DOP-PCR, random primers that also contain universal amplification sequences are used in a first round of annealing and PCR. Then a second
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49/116 round of PCR used to amplify the sequences further with the universal primers. MDA uses 29 polymerase, which is a highly processing, non-specific DNA enzyme that replicates and has been used for the analysis of a single cell. Amplification of the entire genome of a single cell has been used successfully for a variety of applications for a number of years. There are other methods of amplifying DNA from a DNA sample. DNA amplification transforms the initial DNA sample into a DNA sample, which is similar in the set of sequences, but of much larger quantities. In some cases, amplification may not be necessary.
[00128] In some embodiments, DNA can be amplified using universal amplifications, such as WGA or MDA. In some embodiments, DNA can be amplified by specific amplification, for example, using targeted PCR or circularization probes. In some embodiments, the DNA can preferably be enriched using a method of target amplification or a method that results in the partial or total separation of desired DNA from unwanted, such as hybridization capture approaches. In some embodiments, DNA can be amplified using a combination of a universal amplification method and a preferred enrichment method. A more detailed description of some of these methods can be found elsewhere in this document.
[00129] The genetic data of the target individual and / or the related individual, can be transformed from a molecular state to an electronic state by measuring the appropriate genetic material using tools or techniques and made from a group including, but not limited to a: high-throughput genotyping and sequencing microarrays. Some high-throughput sequencing methods include
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Sanger DNA sequencing, pyro-sequencing, the Solexa platform ILLUMINA GENOME ANALYZER or 454 sequencing platform is applied BIOSYSTEM, TRUE single molecule of Helicos SEQUENCING platform, Halcyon electron microscope MOLECULAR method or any other sequencing method. All of these methods of physically transforming the genetic data stored in a DNA sample into a set of genetic data, which is typically stored in a memory device en route to be processed.
[00130] The genetic data of an individual concerned can be measured by analysis of substances made from a group including, but not limited to: the individual's tissue large diploid amounts, one or more diploid cells of the individual, one or more cells haploids of the individual, one or more blastomeres from the individual target genetic material, extra-cellular found in the particular genetic material, extra-cellular from the individual found in maternal blood, from cells of the individual found in maternal blood, from one or more embryos created from a gamete of the related individual, one or more blastomeres made from such an embryo, the extra-cellular genetic material found in the material related to the individual, genetics known to have originated from the related individual and combinations of the themselves.
[00131] In some modalities, the probability that an alleged father is the biological father of a fetus can be calculated. In some modalities, the determination of paternity can be used to make a clinical decision. This knowledge, typically stored as a physical arrangement of matter in a memory device, can then be turned into a report. The report can then be put into practice. For example,
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51/116 clinical decision may be to terminate the pregnancy, alternatively, the clinical decision may be to continue the pregnancy.
[00132] In one embodiment of the present description, any of the methods described here can be modified to allow multiple targets to come from the same individual target, for example, multiple blood drawn from the same pregnant woman. This can improve the accuracy of the model, as multiple genetic measurements can provide more data with which the target genotype can be determined. In one embodiment, a single set of genetic data served as the primary data target, which was reported, and the other serves as data to recheck the primary target genetic data. In one embodiment, a plurality of sets of genetic data, each measured from genetic material taken from the target individual, are considered in parallel, and thus both sets of target genetic data serve to help determine the paternity of the fetus.
[00133] In one embodiment, the method can be used for the purpose of paternity testing. For example, given the SNP-based genotypic information of the mother and a man who may or may not be the genetic father and the genotypic information measured from the mixed sample, it is possible to determine whether the male's genotypic information actually represents the father. genetic profile of the unborn fetus. A simple way to do this is to simply look at the contexts in which the mother is AA and the possible father is AB or BB. In these cases, one can expect to see the contribution half parent (AA | AB) or all (AA | BB) of time, respectively. Bearing in mind the expectation of ADO, it is simple to determine whether or not the fetal SNPs that are observed are related to those of the possible father. Other methods for making a paternity determination are described elsewhere in this document.
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52/116 [00134] In one embodiment of the present description, a pregnant woman would like to determine whether a man is the biological father of her fetus. She goes to her doctor and gives a sample of her blood and she and her husband give samples of their own DNA from smears on the cheek. Genotypes a parental DNA lab researcher using the MDA protocol to amplify the parental DNA and IIlumina Infinium matrices to measure the parents' genetic data on a large number of SNPs. The researcher then spins the blood down, converts the plasma and isolates a free floating DNA sample using size exclusion chromatography. Alternatively, the investigator uses one or more fluorescent antibodies, such as one that is specific for fetal hemoglobin to isolate a fetal nucleated red blood cell. The researcher then converts the isolated or enriched fetal genetic material and amplifies it using a properly designed 70-mer oligonucleotide library in such a way that two ends of each of the oligonucleotides corresponded to the flanking sequences on both sides of a target allele. After the addition of a polymerase, ligase and the appropriate reagents, the oligonucleotides were subjected to circularization and filling in the gaps, capturing the desired allele. The exonuclease was added, inactivated by heat and the products were used directly as a template for PCR amplification. The PCR products were sequenced on a GENOME ILLUMINA analyzer. The sequence reads were used as an input to the PARENTAL SUPPORT ™ method, which then predicted the fetal ploidy state. The method determines that the alleged father is not the biological father of the fetus and calculates the confidence in the determination of 99.98%. A report is generated disclosing both the determination of paternity and the confidence of the determination.
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53/116 [00135] In another modality the woman who is pregnant wants to know if a man is the biological father of her fetus. The obstetrician has a blood collection from the mother and father. The blood is sent to a laboratory, where a technical centrifuge of the maternal sample is used to isolate the plasma and the yellowish cover. The DNA in the yellowish covering and the paternal blood sample are transformed with the amplification and the genetic data encoded in the amplified genetic material is further transformed from data stored in molecular genetics electronically stored genetic data, running the genetic material in a high sequencer. performance to measure parental genotypes. The plasma sample is preferably enriched in a set of loci using a complex hemi-clustered 5,000 target PCR method. The mixture of DNA fragments is prepared in a DNA library suitable for sequencing. The DNA is then sequenced using a high-throughput sequencing method, for example, ILLUMINAGAIIxGENOME ANALYZER. Sequencing transforms the information that is molecularly encoded in the DNA where the information is electronically encoded in the computer hardware. A computer-based technique, which includes currently disclosed modalities, such as PARENTAL SUPPORT®, can be used to determine the fetus' paternity. This may involve calculating, by a computer, the allele counts in the plurality of polymorphic loci from measurements made on the DNA of the enriched sample and determining the probability that the man is the biological father of his fetus. The probability that the alleged father is the biological father of the fetus is determined to be 99.9999%, and the confidence in the determination of paternity is calculated at 99.99%. A report is printed or sent electronically to the pregnant woman's obstetrician, who transmits the determination to the woman. The wife, the husband and the
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54/116 doctor sit down and discuss the report.
[00136] In one embodiment, the raw genetic material of the mother, the father and is transformed by amplification to a quantity of DNA that is similar in sequence, but in greater quantity. Then, by means of a genotyping method, the genotypic data that is encoded by the nucleic acids which turns into genetic measurements that can be stored physically and / or electronically on a memory device, such as those described above. The relevant algorithms that make up the PARENTAL SUPPORT® algorithm, relevant parts that are discussed in detail here, are translated into a computer program, using a programming language. Then, by executing a computer program on the computer hardware, instead of being physically encoded bits and bytes, arranged in a pattern that represents raw measurement data, they become transformed into a pattern that represents a determination of high confidence the paternity of the fetus. The details of this transformation will depend on the data itself and the computer language and hardware system used to perform the method described here. Then, the data that is physically configured to represent a high quality paternity determination of the fetus is transformed into a report that can be sent to a health care professional. This transformation can be done through a printer or a computer monitor. The report can be a hard copy, paper or other suitable medium or it can be electronic. In the case of an electronic report, which can be transmitted, this can be physically stored on a memory device from a location on the computer accessible by the health care professional, but it can also be displayed on the screen in a way that can be read. In the case of a screen, the data
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55/116 can be transformed into a readable format by causing the physical transformation of pixels of the display device. The transformation can be achieved by physically burning electrons to a phosphor screen, by means of an electric charge change that physically changes the transparency of a specific set of pixels on a screen that can be found in front of a substrate, that emits or absorbs photons. This transformation can be achieved by changing the orientation of nanoscale molecules in a liquid crystal, for example, from the nematic or smectic cholesteric phase, to a specific set of pixels. This transformation can be achieved by means of an electric current causing photons to be emitted from a specific set of pixels made from a plurality of light-emitting diodes arranged in a significant pattern. This transformation can be achieved by any other means used to display the information, such as a computer screen or any other form of output or information transmission device. The healthcare professional can then act on the report, so that the data in the report is transformed into an action. The action may be to continue or interrupt the pregnancy, in which case an unborn child becomes a lifeless fetus. Alternatively, a set of genotypic measures can be turned into a report that helps a doctor treat the pregnant patient.
[00137] In some modalities, the methods described here can be used at a very early gestational age, for example, as early as four weeks, from five weeks as early as six weeks, as early as seven weeks, as early as as early as eight weeks as early as nine weeks, as early as 10 weeks, as early as 11 weeks, and as early as 12 weeks.
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56/116 [00138] Any of the modalities disclosed here can be implemented in a digital electronic circuit, integrated circuits, specially designed ASICs (integrated circuits of specific applications), hardware, firmware, software or in combinations thereof. Apparatus of one of the presently disclosed modalities can be implemented in a computer program product tangibly incorporated into a machine-readable storage device for execution by a programmable processor; The method and the steps of the modalities presently described can be performed by a programmable processor, executing an instruction program to execute the functions of the modalities presently disclosed by the operation on input and output generation data. The modalities presently disclosed can be advantageously implemented in one or more computer programs that are executable and / or interpretable in a programmable system including at least one programmable processor, which can be of general or special use, coupled to receive data and instructions from and transmission of data and instructions to a storage system with at least one input device and at least one output device. Each computer program can be implemented in a procedural or object-oriented high-level programming language or in assembly machine language or, if desired, and in any case, the language can be a compiled or interpreted language. A computer program can be implemented in any form, including as an individual program or as a module, subroutine component or other unit suitable for use in a computing environment. A computer program can be deployed to be run or interpreted on one computer or on multiple computers on a website or distributed across multiple
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57/116 local and interconnected by a communication network.
[00139] Computer read storage media, as used herein, refers to physical or tangible storage (as opposed to signs) and includes, without limitation, volatile and non-volatile, removable and non-removable media implemented by any method or technology for storing information, such as tangible computer-readable instructions, data structures, program modules or other data. Computer-readable storage media includes, but is not limited to, RAM, ROM, EPROM, EEPROM, flash memory or other solid-state memory technology, CD-ROM, a DVD or other optical, magnetic, magnetic tape cassettes, magnetic disk storage or other magnetic storage devices or any other physical medium or material that can be used to store tangible information or data or instructions and that can be accessed by a desired computer or processor. Enrichment and Targeting Sequencing [00140] The use of a technique to enrich a DNA sample for a set of target loci followed by sequencing as part of a non-invasive method for the so-called prenatal allele or called ploidy can confer a number of unexpected advantages. In some embodiments of the present description, the method involves measuring genetic data for use with a computerized method, such as PARENTAL SUPPORT® (PS). The end result of some of the modalities is the actionable genetic data of an embryo or fetus. There are many methods that can be used to measure the genetic data of the individual and / or related individuals as part of established methods. In one embodiment, a method for enriching the concentration of a set of target alleles is described here, the method
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58/116 comprising one or more of the following steps: the amplification of the target genetic material, the addition of specific oligonucleotide probe loci, the ligation of specific DNA strands, isolation of desired DNA sets, the removal of unwanted components from a reaction , the detection of certain DNA sequences by hybridization and the detection of the sequence of one or a plurality of DNA strands by DNA sequencing methods. In some cases, the target DNA strands may refer to genetic material, in some cases, the primers may refer, in some cases, it may refer to synthesis sequences or combinations thereof. These steps can be performed in a number of different orders. Given the highly variable nature of molecular biology, it is generally not obvious that the methods and which combinations of steps will perform poorly, well or better in various situations.
[00141] For example, a step of universal DNA amplification before target amplification can confer several advantages, such as eliminating the risk of bottlenecks and reducing allelic bias. The DNA can be mixed with an oligonucleotide probe that can hybridize with both neighboring regions of the target sequence, one on each side. After hybridization, the ends of the probe can be linked by adding a polymerase, a ligation medium and any necessary reagents to allow the probe to circularize. After circularization, an exonuclease can be added to the genetic material to digest non-circularized, followed by detection of the circularized probe. The DNA can be mixed with PCR primers that can hybridize to the two neighboring regions of the target sequence, one on each side. After hybridization, the probe ends can be linked by adding a polymerase, a ligation medium and any reagents needed to complete the
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59/116 PCR amplification. Amplified or non-amplified DNA can be targeted by hybrid capture probes that target a set of loci, after hybridization, the probe can be identified and separated from the mixture to provide a mixture of DNA, which is enriched in target sequences.
[00142] In some modalities the detection of the target genetic material can be done in a multiplex way. The number of genetic target sequences that can be executed in parallel can vary from one to ten, ten to one hundred, 100-1000, 1000-10000, 10000-100000, 1000001.000.000 or from 1 to 10 million. Note that the prior art includes the description of successful PCR reactions involving multiplex pools of up to about 50 or 100 primers and no more. Previous attempts to multiplex more than 100 primers per pool have resulted in significant problems with undesirable side reactions, such as the formation of primer dimers.
[00143] In some embodiments, this method can be used for the genotyping of a single cell, a small number of cells, 2-5 cells, from six to ten cells, cells from ten to twenty, twenty to fifty cells, fifty to a hundred cells, a hundred to a thousand cells or a small amount of extracellular DNA, for example, from one to ten picograms, 10-100 picograms, from a hundred picograms to nanograms one, from one to ten nanograms, 10-100 nanograms or from one hundred nanograms to one microgram.
[00144] The use of a method to reach certain loci followed by sequencing as part of a method for the so-called allele or called ploidy can confer a number of unexpected advantages. Some methods by which DNA can be targeted or preferably enriched, include the use of circularization probes, inverted linked probes (lips, MIPs), capture by hybridization methods such as SureSelet and targeted PCR or
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60/116 PCR-mediated ligation amplification strategies.
[00145] In some embodiments, a method of the present disclosure involves measuring genetic data for use with a computerized method, such as PARENTAL SUPPORT® (PS). PARENTAL SUPPORT® is a computer-based approach to manipulating genetic data, aspects of which are described below. The end result of some of the modalities is the actionable genetic data of an embryo or fetus followed by a clinical decision based on the actionable data. The algorithms behind the PS method take the genetic data of measuring the individual target, often an embryo or fetus, the genetic data and measured from related individuals and are able to increase the accuracy with which the genetic condition of the individual target is known. In one embodiment, genetic measurement data is used in the context of making paternity determinations during prenatal genetic diagnosis. There are many methods that can be used to measure the genetic data of the individual and / or the related individuals in the contexts mentioned above. The different methods of understanding a number of steps, the steps that often involve amplifying the genetic material, adding oligonucleotide probes, ligating certain DNA strands, isolating desired DNA pools, removing unwanted components of a reaction, the detection of certain DNA sequences by hybridization, detection of the sequence of one or a plurality of DNA strands by DNA sequencing methods. In some cases, the target DNA strands may refer to genetic material, in some cases, the primers may refer, in some cases, it may refer to synthesis sequences or combinations thereof. These steps can be performed in a number of different orders. Given
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61/116 the highly variable nature of molecular biology, which is generally not obvious that the methods and which combinations of steps, will perform poorly, well or better in various situations.
[00146] Some modalities of the present description involve the use of inverted Linked probes (GPI), which were previously described in the literature. GPI is a generic term intended to encompass technologies that involve the creation of a circular DNA molecule, in which the probes are designed to hybridize to the target DNA region on both sides of a target allele, such that the addition of polymerases appropriate and / or ligases and the appropriate conditions, buffers and other reagents, will complete the complementary inverted DNA region across the target allele to create a circular loop of DNA, which captures the information found in the targeted allele. GPI can also be called pre-circularized probes, pre-circular probes or circularization probes. The lips probe can be a linear DNA molecule between 50 and 500 nucleotides in length and in a modality between 70 and 100 nucleotides in length, in some embodiments, it can be longer or shorter than that described here. Other modalities of the present description involve different incarnations of edge technology, such as PADLOCK probes and Molecular Inversion Probes (MIPs).
[00147] A method for targeting specific sites for sequencing is to synthesize probes in which the 3 'and 5' ends of the probes bind to the target DNA at sites adjacent to and on both sides of the target region, in an inverted manner. such that the addition of DNA polymerase and DNA ligase results in extension of the 3 'end, the addition of bases to the single-stranded probe that are complementary to the target molecule (gap-fill), followed by binding of the new 3' end to the 5 'end of the original probe resulting in a circular DNA molecule, which
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62/116 can subsequently be isolated from background DNA. The ends of the probe are designed to flank the target region of interest. One aspect of this approach is commonly called MIPS and has been used in conjunction with parent technologies to determine the nature of the filled sequence [00148] PCR-mediated binding is a PCR method used to enrich, preferably a DNA sample by amplification of one or a plurality of loci of a DNA mixture, the method comprising: obtaining a set of primer pairs, where each primer in the pair contains a specific sequence target and a non-target sequence, wherein the target sequence specific was designed to hybridize with a target region, one upstream and one downstream of the polymorphic site; polymerization of DNA from the 3-prime end of the upstream primer to fill the single-strand region between it and the 5-prime end of the downstream primer with the complementary nucleotides of the target molecule; ligation of the last polymerized base of the primer upstream of the 5-prime base adjacent downstream of the primer and amplification of only polymerized and ligated, using the non-target sequence molecules contained in the 5-prime end of the upstream primer and the end 3 next to the downstream initiator. The pairs of primers for different targets can be mixed in the same reaction. The non-target sequences serve as universal sequences in such a way that all pairs of primers that have been successfully polymerized and ligated can be amplified with a single pair of amplification primers.
[00149] In one embodiment, a DNA sample can be preferably enriched using a hybridization capture method. Some examples of commercial capture of hybridization technologies include SureSelet from Agilent and TRUSEQ from ILLUMINA.
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In hybridization capture a set of oligonucleotides that is complementary or almost complementary to the desired target sequences is allowed to hybridize to a mixture of DNA and then physically separated from the mixture. Once the desired sequences have hybridized to the targeting oligonucleotides, the effect of physically removing the target oligonucleotides is also to remove the target sequences. Once the hybridized oligos are removed, they can be heated above their melting temperature and they can be amplified. Some ways to physically remove the target oligonucleotides is by covalently bonding the target oligos to a solid support, for example, a magnetic sphere or a chip. Another way to physically remove targeting oligonucleotides is to covalently attach them to a molecular moiety with a strong affinity for another molecular moiety. An example of such a molecular pair is biotin and streptavidin, as used in SureSelet. Once the target sequences can be covalently linked to a biotin molecule, and, after hybridization, with a solid streptavidin support can be used to pull down the biotinylated oligonucleotides, which hybridize to the target sequences.
[00150] In some embodiments, PCR can be used to target specific locations in the genome. In plasma samples, the original DNA is highly fragmented (typically less than 500 bp, with an average length of less than 200 bp). In PCR, both forward and reverse primers must merge with the same fragment, to allow amplification. Therefore, if the fragments are short, the PCR assays should amplify relatively short regions as well. PCR assays can be generated in large numbers, however, the interactions between different PCR assays make it difficult to multiplex them beyond about one hundred assays. Various approaches
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64/116 complex molecules can be used to increase the multiplex level, but it can still be limited to less than 100, perhaps 200, 500 or possibly by reaction assays. Samples with large amounts of DNA can be divided between multiple subreactions and then recombined before sequencing. For samples where either the global sample or some subpopulation of DNA molecules is limited, dividing the sample does not introduce statistical noise. In one embodiment, a small or limited amount of DNA can refer to less than 10 pg, between 10 and 100 pg, between 100 pg and 1 ng, between 1 and 10 ng or between 10 and 100 ng. Note that although this method is particularly useful in small amounts of DNA, where other methods that involve splitting into multiple pools can cause significant problems related to the introduced stochastic noise, this method still offers the advantage of minimizing bias when it is performed on samples of any amount of DNA. In these situations, a universal pre-amplification step can be used to increase the overall sample volume. Ideally, this pre-amplification step should not significantly alter the allelic distributions.
[00151] In general, to perform target sequencing of multiple targets (n) of a sample (greater than 50, greater than 100, greater than 500 or more than 1000), one can divide the sample into a certain number parallel reactions that amplify one or a smaller number of individual targets. This was performed on plates of several PCR pools or can be done on commercial platforms such as the Fluidigm Access matrix (48 reactions per sample on microfluidic chips) or DROP PCR by Rain Dance TECHNOLOGY (100s to a few thousand targets). Unfortunately, these e-pool separation methods are problematic for samples with a limited amount of DNA, as there are often not enough copies of the
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65/116 genome to ensure that there is a copy of each of the regions of the genome in each pool. This is an especially serious problem when polymorphic loci are segmented and the relative proportions of alleles in polymorphic loci are necessary, as the noise introduced by stochastic division and sharing will cause very accurate measurements of the proportions of the alleles that were present in the original DNA sample. . Described here is an effective and efficient method for amplifying many PCR reactions that apply to cases where only a limited amount of DNA is available. In one embodiment, the method can be applied for the analysis of individual cells, body fluids, mixtures of DNA, such as free floating DNA in maternal plasma, biopsies, environmental and / or forensic samples.
[00152] In one embodiment, the target sequence can involve one, a plurality or all of the following steps, a) Generate and universally amplify a library of adapter sequences at both ends of the DNA fragments. b) Divide into multiple reactions after amplifying the library, c) Perform about 100-plex, about 1000-plex or about 10,000 complex amplification of selected targets, using a specific target Primer per-target primer and a specific tag primer . d) Perform a second amplification of this product using specific target Reverse primers and one (or more) of specific primers for a universal brand, which was introduced as part of the specific forward primers target in the first round, e) Divide the product into multiple aliquots and enlarge subpools of targets in individual reactions (eg 50 to 500-plex, although this can be used all the way up to singleplex. f) Pool products of parallel subpools reactions. During these amplifications, initiators can perform compatible tag sequencing (full or partial length) in
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66/116 so that products can be sequenced.
[00153] In one embodiment, it is possible to mitigate potential losses in the subsequent steps, amplifying all or a fraction of the DNA of the free sample of original cells (cfDNA). Several methods are available to amplify all the genetic material in a sample, increasing the amount available for downstream processes. In one embodiment, the LM-PCR-mediated ligation (PCR) of DNA fragments are amplified by PCR after ligation of either, two different adapters or different different adapters. In one embodiment, multiple displacement amplification (MDA) Phi-29 polymerase is used to amplify all DNA isothermally. In DOP-PCR and variations, random initiation is used to amplify the original material DNA. Each method has certain characteristics, such as the uniformity of amplification in all regions of the genome represented, the efficiency of capture and amplification of original DNA and the amplification performance, depending on the length of the fragment.
[00154] Results of the traditionally designed PCR assay at significant losses of distinct fetal molecules, but the losses can be reduced by designing very short PCR assays, the so-called mini-PCR assays. Fetal cfDNA in maternal blood is highly fragmented and fragment sizes are distributed in approximately a Gaussian form with an average of about 160 bp, a standard deviation of about 15 bp, a minimum size of about 100 bp and a maximum size of about 220 bp. The distribution of the starting and ending positions of the fragments in relation to specific polymorphisms, although not necessarily random, varies widely between individual targets and between all the block targets and the polymorphic site of a
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67/116 a specific target locus can occupy any position, from the beginning to the end among the various fragments from that location. Note that the term mini-PCR can also refer to normal PCR, without additional restrictions or limitations.
[00155] During PCR, amplification will only occur from template DNA fragments comprising both forward and reverse primer sites. Because fetal cfDNA fragments are short, the probability of both primer sites, the probability of a fetal fragment of length L being present comprising both the front and reverse primer sites being present, is the ratio of the length of the amplification product to the length fragment. Under ideal conditions, tests where the amplified product is 45, 50, 55, 60, 65 or 70 bp will successfully amplify from about 72%, 69%, 66%, 63%, 59% or 56% , respectively, of available model fragment molecules. The length of the amplified product is the distance between the 5-prime ends of the forward and reverse initiation sites. Amplicon length that is shorter than normally used by those known in the art can result in more efficient measurements of the desired polymorphic loci by requiring only small sequence reads. In one embodiment, a substantial fraction of the amplification products must be less than 100 bp, less than 90 bp, less than 80 bp, less than 70 bp, less than 65 bp, less than 60 bp, less than 55 bp, less than 50 bp or less than 45 bp.
[00156] Note that in the methods known in the prior art, short tests, such as those described here, are generally avoided because they are not necessary and impose considerable restriction on the design of primers by limiting the length of the initiator, annealing characteristics and the distance between the forward and reverse primer.
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68/116 [00157] Multiplex PCR may involve a single round of PCR, in which all targets are amplified or may involve a round of PCR, followed by one or more cycles of nested PCR or some variant of nested PCR. The PCR consists of a subsequent round or rounds of PCR amplification using one or more of the new primers that internally bind at least one base pair to the primers used in the previous round. PCR reduces the number of spurious amplification targets per amplification, in subsequent reactions, only amplification products from the previous one that have the correct internal sequence. Reducing spurious amplification targets increases the number of useful measurements that can be obtained, especially in sequencing. PCR generally involves designing completely internal primers for the binding sites of previous primers, necessarily increasing the minimum DNA segment size needed for amplification. For samples, such as maternal cfDNAplasma, where DNA is highly fragmented, the larger the size of the assay reduces the number of distinct cfDNA molecules from which a measurement can be obtained. In one embodiment, to compensate for this effect, a partial settlement approach can be used in which one or both of the second round primers overlap the first binding sites that extend a certain number of bases internally to achieve additional specificity while minimally increasing in the total test size.
[00158] In one embodiment, a set of multiplex PCR assays were designed to amplify potentially heterozygous SNP or other polymorphic or non-polymorphic loci on one or more chromosomes and these assays are used in a single DNA amplification reaction. The number of PCR assays can be between
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69/116 and 200 PCR assays, between 200 and 1000 PCR, between 1000 and 5000 PCR or between 5,000 and 20,000 PCR assays (50 to 200-Plex, 200 to 1000-Plex, 1,000 to 5000-plex, 5,000 to 20,000 complex, more than 20,000 complex respectively).
[00159] In one embodiment, a 100-plex to 500 complex, 500plex to 1000-plex, 1000-plex to 2000-plex, 2000-plex to 5000plex, 5000-plex to 10,000 complex, 10,000 to 20,000 plex complex, 20,000 -plex to 50,000-plex or 50,000-plex to 100,000-plex PCR pool assay is created in such a way that forward and reverse primers have tails corresponding to the required forward and reverse sequences required by a high throughput transfer instrument, such as HiSeq, GAIIX or available from MISEQ ILLUMINA. In addition, the included 5-primer for sequencing tails is an additional sequence that can be used as a start site for a subsequent PCR to add barcode nucleotide sequences to the amplicons, allowing for multiplex sequencing of multiple samples from one single track high-performance sequencing instrument. In one embodiment, a 10,000 PCR assay pool complex is created in such a way that the reverse primers have tails corresponding to the required inverted sequences required by a high-throughput sequencing instrument. After amplification with the first 10,000-Plex assay, subsequent CPR amplification can be performed using another set of 10,000 plex having forward nodes partially nested (eg 6 nested bases) for all targets and a reverse primer corresponding to the tail reverse sequencing included in the first round. This subsequent round of amplification partially nested with only one specific target primer and one universal primer, limits the required size of the assay, reducing noise from
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70/116 sampling, but greatly reduces the number of spurious amplification products. The sequencing markers can be added to attached connection adapters and / or as part of PCR probes, such that the marking is part of the final amplification product.
[00160] Fetal fraction affects the performance of the assay, it is more difficult to determine the correct paternity in samples with a smaller fetal fraction. There are a number of ways to enrich the fetal DNA fraction in maternal plasma. Fetal fraction can be increased by the method previously described LM-PCR has already been discussed, as well as by a specific removal of long mathematical fragments. In the modality, the longest fragments are removed using size selection techniques. In one embodiment, prior to multiplex PCR amplification at the target loci, an additional multiplex PCR reaction can be performed to selectively remove fragments of length, and largely maternal, corresponding to the target loci in the subsequent multiplex PCR. The primers were designed to pair a site at a greater distance than the polymorphism is expected to be present between fetal cell-free DNA fragments. These primers can be used in a multiplex PCR reaction cycle before the multiplex PCR of the target polymorphic loci. These distal primers are labeled with a molecule or portion that can allow selective recognition of the labeled parts of DNA. In one embodiment, these DNA molecules can be covalently modified with a biotin molecule, which allows removal of newly formed double-stranded DNA comprising these primers after a PCR cycle. Double-stranded DNA formed during the first round is likely maternal in origin. The removal of the hybrid material can be used by performing spheres of
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71/116 magnetic streptavidin. There are other dialing methods that can work just as well. In one embodiment, size selection methods can be used to enrich the DNA sample of shorter strands, for example, those with less than about 800 bp, less than about 500 bp or less than about 300 bp . Amplification of short fragments can then proceed as usual.
[00161] In some embodiments, the target DNA may originate from individual cells, from DNA samples consisting of less than one copy of the target genome, from small amounts of DNA, from DNA of mixed origin (for example, plasma pregnancy: placental and maternal DNA; patient plasma cancer and tumors: mixture of healthy DNA and cancer, transplantation, etc.), from other body fluids, from cultures of cells, from culture supernatants, from forensic DNA samples, from old DNA samples (eg, insects trapped in amber), from other DNA samples and combinations thereof.
[00162] In some embodiments, the methods described here can be used to amplify and / or detect SN Ps, simple tandem repeats (STR), copy number, nucleotide methylation, mRNA levels, other types of expression levels of RNA, other genetic and / or epigenetic characteristics. The mini-PCR methods described here can be used together with next generation sequencing, which can be used with other downstream methods, such as microarrays, counting by digital PCR, real-time PCR, mass spectrometry analysis, etc. [00163] In some embodiments, the mini-PCR amplification methods described here can be used as part of a method for the exact quantification of minority populations. He can be
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72/116 used for absolute peak quantification using calibrators. It can be used for mutation / minor allele quantification through very deep sequencing and can be performed in a highly multiplex manner. It can be used for standard paternity tests and identity of relatives or ancestors, on humans, animals, plants or other creatures. It can be used for forensic testing. It can be used for rapid genotyping and copy number (CN) analysis on any type of material, for example, amniotic fluid and CVS, sperm, product of conception (POC). It can be used for the analysis of a single cell, such as genotyping samples subjected to biopsy from embryos. It can be used for rapid analysis of embryos (in less than one, one or two days of the biopsy) by targeted sequencing using min-PCR. [00164] Multiplex PCR can often result in the production of a very high proportion of the DNA product resulting from non-productive side reactions, such as the formation of a primer dimer. In one embodiment, specific primers that are more likely to cause non-productive side reactions can be removed from the primer library to give a primer library that will result in a higher proportion of amplified DNA that maps to the genome. The step of removing problematic primers, that is, those which are particularly susceptible to firm dimers, has unexpectedly enabled extremely high levels of multiplex PCR formation for further analysis by sequencing. In systems such as sequencing, where performance degrades significantly by initiator dimers and / or other injury products, greater than 10, greater than 50 and greater than 100 times greater than the other multiplex multiplex formation described has been achieved. Note that it is opposed to investigating detection methods with bases, for example,
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73/116 microarrays, TaqMan, PCR, etc., where an excess of primer dimers will not appreciably affect the result. It should also be noted that the general belief in the technique is that PCR for sequencing multiplex formation is limited to about 100 tests in the same pool.
[00165] There are a number of ways to choose primers for a library in which the amount of non-mapping primer dimers or other Harm primer products is minimized. Empirical data indicates that a small number of 'bad' primers are responsible for a large number of non-mapped dimer side reactions. Removing these bad primers can increase the percentage of sequence reads that map to the target loci. One way to identify bad primers is to look at the DNA sequencing data that has been amplified by specific amplification; those primer dimers that are seen most often can be removed to give a primer library that is significantly less likely to result in DNA by-product that is not mapped to the genome. There are also publicly available programs that can calculate the binding energy of various combinations of primers and removing those with the highest binding energy will also give a booklet library that is significantly less likely to result in DNA product side that is not mapped to the genome.
[00166] Note that there are other methods for determining which PCR probes are likely to form dimers. In one embodiment, analyzing a set of DNA that has been amplified using a non-optimized set of primers may be sufficient to determine problematic primers. For example, the analysis can be done using sequencing and those dimers that are
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74/116 present in greater numbers are determined as those that are most likely to form dimers and can be removed.
[00167] To select the target locations, one can start with a group of alleged pair of initiators projects and create a thermodynamic model of potentially adverse interactions between the pairs of initiators, and then use the model to eliminate the projects that are incompatible with the other projects in the pool.
[00168] There are many workflows that are possible when performing PCR, some typical flows for the methods described here are described. The steps described in this document are not intended to exclude other possible steps and it does not mean that any of the steps described here are necessary for the method to work properly. A large number of parameter variations or other modifications are known in the literature and can be made without affecting the essence of the invention. A specific generalized workflow is given below, followed by a number of possible variants. The variants typically refer to possible secondary PCR reactions, for example, different types of settlement that can be performed (step 3). It is important to note that the variants can be made at different times or in different orders as explicitly described here.
1. The DNA in the sample may have ligation adapters, often referred to as the attachment adapter libraries (LTs) tags, attached, where the ligation adapters contain a universal initiation sequence, followed by universal amplification . In one embodiment, this can be done using a standard protocol designed to create sequencing libraries after fragmentation. In one embodiment, the DNA sample can be blunt, and then an A can be added at the end 3 A Y-adapter with a T-overhang can be added
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75/116 added and linked. In some embodiments, other cohesive ends can be used in addition to a shoulder A or T. In some embodiments, other adapters can be added, for example coiled connection adapters. In some embodiments, adapters may have marking designed for PCR amplification.
2. Target specific amplification (STA): Pre-amplification from hundreds of thousands to tens of thousands and even hundreds of thousands of targets can be multiplexed in one reaction. STA is typically run from 10 to 30 cycles, although it can be run from 5 to 40 cycles, from 2 to 50 cycles and even from 1 to 100 cycles. The primers can be tied, for example, for a simple or sequencing workflow to avoid a large proportion of dimers. Note that typically, dimers from both primers that carry the same tag will not be efficiently amplified or sequenced. In some modalities, between 1 and 10 cycles of PCR can be performed, in some modalities between 10 and 20 cycles of PCR can be performed, in some modalities between 20 and 30 cycles of PCR can be performed, in some modalities between 30 and 40 PCR cycles can be performed, in some modalities more than 40 PCR cycles can be performed. The amplification can be a linear amplification. The number of PCR cycles can be optimized to result in an optimal profile reading depth (DOR). Different PAIN profiles may be desirable for different purposes. In some modalities, a more uniform distribution of all tests between readings is desirable, if the DOR is too small for some tests, the stochastic noise may be too high for the data to be useful, while, if the reading depth too high, the marginal utility of each additional reading is relatively small.
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76/116 [00169] Primer can improve fragmented DNA detection from universally labeled libraries. If the library tag and the primer-tails contain a homologous sequence, hybridization can be improved (for example, the melting temperature (TM) is reduced) and the primers can be extended, if only a portion of the primary target sequence is in the sample's DNA fragment. In some embodiments, 13 or more base pairs can be used for a specific target. In some embodiments, 10 to 12 base pairs can be used for a specific target. In some embodiments, 8-9 base pairs for a specific target can be used. In some embodiments, 6 to 7 base pairs can be used for a specific target. In some embodiments, STA can be performed on pre-amplified DNA, for example, MDA, RCA, others amplification of the entire genome or adapter mediated universal PCR. In some modalities, STA can be performed on samples that are enriched or exhausted from certain sequences and populations, for example, selection by size, target capture, directed degradation.
3. In some modalities, it is possible to perform secondary multiplex PCR or primer extension reactions to increase specificity and reduce undesirable products. For example, full of settlement, semi-settlement, hemisettlement and / or subdivision in parallel reactions of smaller test pools are all techniques that can be used to increase specificity. Experiments have shown that splitting a sample into three 400 complex reactions resulted in product DNA with greater specificity than a 1200-plex reaction with exactly the same primers. Similarly, experiments have shown that splitting a sample into four reactions of 2,400 complexes resulted in a DNA product with greater specificity
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77/116 than a 9600-Plex reaction with exactly the same primers. In one embodiment, it is possible to use specific targeting and targeting initiators of the same direction and opposites.
4. In some modalities, it is possible to amplify a sample of DNA (dilution, purified or otherwise), produced by a STA reaction using specific primers for labeling and universal amplification, that is, to amplify many or all preamplified and marked targets . The initiators may contain additional functional strings, for example, bar codes or a complete adapter string required for sequencing on a high throughput sequencing platform.
[00170] These methods can be used to analyze any DNA sample and are especially useful when the DNA sample is particularly small or when it is a DNA sample, where the DNA more than originates from an individual, such as in the case of maternal plasma. These methods can be used on DNA samples, such as a single or small number of cells, genomic DNA, plasma DNA, amplified plasma libraries, amplified apoptotic supernatant libraries or other mixed DNA samples. In one embodiment, these methods can be used in the event that cells of different genetic makeup may be present in an individual, such as cancer or with transplants.
[00171] In some modalities, multiplex PCR amplification may involve the use of several types of settlement protocol, for example, mini-semi-nested-PCR, fully embedded mini-PCR, heminested mini-PCR, triple mini heminested-PCR unilateral nested mini-PCR, on one side or inverse semi-nested mini-PCR mini-PCR.
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78/116
Diagnostic Box [00172] In one embodiment, the present description includes a diagnostic box, which is capable of, in part or completely realizing aspects of the methods described in the present description. In one embodiment, the diagnostic box can be located in a doctor's office, a hospital laboratory, or in any appropriate location reasonably proximal to the patient's point of care. The cashier may be able to perform aspects of the method in a fully automated manner, or the cashier may require one or a number of steps to be completed manually by a technician. In one embodiment, the box may be able to analyze the genotypic data measured in maternal plasma. In one embodiment, the box can be linked to means for transmitting the measured genotypic data using the diagnostic box to an external calculation facility, which can then analyze the genotypic data and possibly also generate a report. The diagnostic box may include a robotic unit that is capable of transferring aqueous or liquid samples from one container to another. It can comprise a number of reagents, both solid and liquid. It can comprise a high throughput sequencer. She can understand a computer.
Primer Kit [00173] In some embodiments, a kit can be formulated which comprises a plurality of primers designed to achieve the methods described in the present description. Primers can be outer front and reverse primers, inner front and reverse primers as described here, they can be primers that are designed to have a low binding affinity to other primers in the kit as described in the primer design section, could be hybrid capture probes or pre-circularized probes
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79/116 as described in the relevant sections or some combination thereof. In one embodiment, a kit can be formulated for determining a ploidy state of a target chromosome in a gestating fetus designed for use with the methods described here, the kit comprises a plurality of forward internal primers and, optionally , a plurality of interior reverse primers and optionally exterior forward primers and exterior reverse primers, where each primer is designed to hybridize to the DNA region immediately upstream and / or downstream of one of the polymorphic sites on the target chromosome and optionally additional chromosomes. In one embodiment, the starter kit can be used in combination with the diagnostic box described elsewhere in this document.
Maximum Probability Estimates [00174] Many methods known in the art for detecting the presence or absence of a phenotype or genotype, for example, a chromosomal abnormality, a medical condition or a paternity relationship involve the use of a single rejection test. of the hypothesis, if a metric that is directly ralted or related to the condition is measured and if the metric is on one side of a certain threshold, the condition is determined to be present, whereas if the metric falls on the other side threshold, the condition is determined to be absent. A single hypothesis rejection test only looks at the null distribution when deciding between the null and alternative hypotheses. Without taking into account the alternative distribution, it is not possible to estimate the probability of each hypothesis given the observed data and, therefore, it is not possible to calculate the confidence in the call. Thus, with a single hypothesis rejection test, a yes or no answer is obtained, without an estimate of the confidence associated with the specific case.
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80/116 [00175] In some modalities, the method described here is able to detect the presence or absence of genotype or phenotype, for example, a chromosomal abnormality, a pathology or a paternity relationship, using a method of maximum probability. This is a substantial improvement over the method of using a single technique to reject the hypothesis that the threshold for calling the absence or presence of the disease can be modified as appropriate for each case. This is particularly relevant for diagnostic techniques that aim to determine the paternity of a fetus in gestation from genetic data available from the mixture of fetal and maternal DNA present in free-floating DNA found in maternal plasma. The maximum probability estimation method can use the allele distributions associated with each hypothesis to estimate the probability of the conditioned data in each hypothesis. These conditional probabilities can then be converted to a so-called hypothesis and confidence. Likewise, a method of maximum posterior estimation uses the same conditional probabilities as the maximum probability estimate, but it also incorporates prior populations when choosing the best hypothesis and determining confidence.
[00176] Therefore, the use of a maximum probability estimation technique (MLE) or closely related to the maximum of a posterior technique (MAP) gives two advantages, firstly, it increases the possibility of a correct call and also allows a confidence to be calculated for each call. In one embodiment, the selection of the so-called paternity corresponding to the hypothesis with the highest probability is performed using maximum probability estimates or a maximum a posteriori estimate. In one embodiment, a method is described to determine the paternity of an unborn fetus that involves taking any method
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81/116 currently known in the art that uses a single technique to reject the hypothesis of reformulating those using an MLE or PAM technique
A Method for Determining Paternity [00177] ffDNA is typically present in the bottom fraction in a mixture with maternal DNA. In some modalities, the mother knew the genotype or the maternal genotype can be measured or inferred. Typically, the fraction of fetal DNA in maternal plasma is between 2 and 20%, although under different conditions this percentage can vary between about 0.01% to about 50%. In one embodiment, a microarray technique or another that gives intensity data on an allele basis can be used to measure maternal plasma. In one embodiment, sequencing can be used to measure the DNA contained in maternal plasma. In these cases, the measurement of the intensity of the allele or sequence reading count in a particular allele is a sum of the maternal and fetal signals. Assuming that the ratio of the child mixture to the matrix DNA is r to 1, the relative number of alleles at the locus consists of two alleles of the mother and 2r alleles of the child. In some personification, the loci comprise single nucleotide polymorphisms. Table 1 shows the relative number of each allele in the mix for a selection of informative parent contexts.
Table 1: Number of alleles per context B in mixture Parental context A in mixture AA | AA 2 + 2r 0 AA | BB 2 + r r AB | AA 1 + 2r 1 AA | AB 2 + 2 or 2 + r 0 or r BB | AA r 2 + r BB | BB 0 2 + 2r
[00178] Note that the choice of the four contexts described above
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82/116 as being informative is not intended to be inclusive of all contexts that may be informative. Any combination of contexts can be used and there is a significant amount of information that can be found in the genotypic measurements of any context.
[00179] Even in the presence of a significant child allele dropout rate, there can be a clear distinction between the signal when an allele is present and in which an allele of the signal is not present. For example, consider allele A measurements of SNPs in BB context pairs | BB and BB | AA and the measurements of B alleles of SNPs in the context of AA pairs | AA and AA | BB. In each case, there should be no sign present in the first context and there must be no sign present in the second direction, where the child's alleles are not eliminated. However, if the alleged parent is incorrect, there will sometimes be a signal present in both contexts. Thus, the distribution of SNP measurements must be different, depending on whether the alleged parent is correct or not.
[00180] The difference will typically be more noticeable at the high end of the distribution signal, as these will be the SNPs where there is a greater likelihood of having DNA contributions from the child. This difference can be seen by comparing high percentage values of the distributions of the SNP measurements. Examples of possible percentile methods are closest position, linear interpolation between closest rows and weighted percentage.
[00181] For example, define X1 as the set of an allele SNP measurements in the BB | BB and X2 as the set of measures of the allele A in BB context | AA, of all chromosomes. If the supposed parent is correct, then the 99th percentile value for X1 will be significantly less than the 99th percentile value for X2. If the
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83/116 supposed parent is incorrect, the 99th percentile values of the two distributions will be closer together. Note that any percentage can be used equally well, for example, the 95th percentile, 90th percentile, 80th percentile or 85th percentile. In one embodiment, for a particular measurement channel, X1 can be defined as the measurements from the context, with no signal and X2 can be defined as measurements from the context in which the mother and father are both homozygous and only the father alleles provide a signal (inherited by the child).
[00182] Define p1 as the 99 (or, 90 95, etc.) percentile of the X1 data and P2 as the 99 (or, 90 95, etc.) percentile of the X2 data. Define the t-test statistic as P1 / P2. Note that the other functions of P1 and P2, which demonstrate the difference in values, can be used equally well. The value of t can vary depending on how much child DNA is present in the sample, which is not known. Therefore, the classification thresholds for t cannot be calculated a priori.
[00183] The t-test statistic for a single sample can be compared with a distribution generated from the genotypes of many individuals who are known not to be the father, using the following procedure. Assume that the genotype of a large set of unrelated individuals is available.
1. For each independent individual, assume that he is the father and calculate the value of the t-test statistic.
2. Let us be the set of measures t of unrelated men. Set up a Tu distribution. This is the distribution of t for the particular sample, under the null hypothesis. The null hypothesis is that genotypes do not come from the child's father present in the sample. The Pu (t) distribution could be the maximum likelihood adjustment or method of the moments to fit a known distribution, for
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84/116 example, a Gaussian distribution, a kemal density adjustment using a kemel function, for example, Gaussian, box, triangle, etc. or any other suitable method of distribution.
3. Consider the alleged father's genotypes and calculate the corresponding tc statistical test.
4. The true parent must result in a lower value of t than an independent individual. The probability of a parent tc independent production or a more peripheral value is the accumulated density function of Pu evaluated in tc. Thus, the p-p-value for rejecting the null hypothesis is given by the following:
p = í ^Ρ “< >
[00184] If p is less than a threshold of significance α then the hypothesis that the father is supposed to be an unrelated individual can be rejected with α meaning. If p is greater than α then the null hypothesis cannot be rejected and the alleged father may not be related to the child is present in the sample. The significance limit α defines the probability that an unrelated individual could be classified as the correct parent. For example, with an α threshold of 0.01, one percent of unrelated individuals could be identified as the correct parent.
[00185] Various methods can be used for combining data from allele channels A and B. For example, two simple methods that require the p-value of all channels to be below a threshold or to require the p-value channel is less than the threshold.
[00186] In some modalities, the paternity test method assumes that the child's DNA is present in a concentration sufficient to distinguish between the SNPs that have or have no sign of the child. In the absence of sufficient child DNA concentration, this method may report an incorrect parent because expected hereditary alleles are not measured in the
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85/116 maternal plasma. In one embodiment, a method is described that can confirm the presence of sufficient child DNA before applying the paternity test. Confirmation of the child's presence is based on a test statistic calculation, which is proportional to the child's DNA concentration, but does not require parent genotypes. If the test statistic is above the required threshold, then the DNA concentration for the child is sufficient to perform the paternity test.
[00187] Consider the set of SNPs (from all combined chromosomes) where the mother genotype is AA and channel B is measured. A signal is expected only in the subset of SNPs where the genotype contains a child B, but these SNPs cannot be identified, a priori, without the parent genotypes, which are not available. Instead, consider populations SNP frequencies {fi}, where fi is the sample mean number of Bs in the SNP i genotype, based on a large population sample. Note that more than SNPs where the matrix is the AA genotype will have a fi less than 0.5, but the distribution of music in these SNPs extends almost to one. Consider two sets of SNPs, S1 and S2, where S1 = {i: fi <TL} and S2 = {i: fi> TH}. The thresholds TL and TH are defined so that very few SNPs in S1 should have a B and many SNPs in S2 should have a B and each set has a sufficient population. In one embodiment, the algorithm uses TL = 0.05 and TH = 0.7, while the other values of TL and TH may work equally well or better. Let yi be the measure B channel of SNP i, Y1 = {yi: i ε S1} and Y2 = {yi: i ε S2}. The distributions of Y1 and Y2 will be very similar, as most SNPs in both distributions will have no signal. However, a nontrivial number of SNPs in S2 is expected to have a child signal and very few SNPs in S1 are expected to have a child signal. Therefore, the tail of Y2 must extend to a greater extent than the tail of Y1. Let's pt be
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86/116 a percentage close to 1, for example, the 99th percentile. In the presence of sufficient child DNA, the pt Y2 percentile should be significantly higher than the P1 percentile of Y1. Thus, the test statistic s can be defined as follows.
s = percentile (Y2; pt) - percentile (Y <pt) [00188] In one embodiment, the statistical test can be normalized by a variety of methods to try to account for differences in amplification between matrices. In one embodiment, normalization can be done on a chromosome basis. In one embodiment, normalization can be done on a per matrix basis. In one embodiment, normalization can be done on a limited basis by sequencing.
[00189] The following calculation shows how the limits of TL and TH are able to distinguish the effect of the child's DNA, in particular maternal sample, based on approximate numbers of SNPs and dropout rates. Table 3 shows some data [00190] In one embodiment, the method involves the following assumptions:
[00191] Population frequencies are calculated from a large population data set, for example, more than 500 individuals, more than 1,000 people, more than 5,000 people or more than 20000 people and the number of SNPs in each context deals with an example mother and father.
[00192] There are no SNPs where the mother is AA and the father is BB (in reality, these are about 8 percent of the mother AA SNPs) [00193] Half of the SNPs where the father has resulted B in child B. [00194 ] Child abandonment rate is 90 percent.
[00195] Measurements with a child signal will be greater than measurements without a child signal [00196] Table 3 shows some data from a case of
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87/116 paternity in particular using the method described with the above parameters. The 98th percentile measurement from S1 is not expected to include any signal with child SNPs present. The measurement of the 98th percentile of S2 should include about 50 SNPs with a child signal present. The difference between the two must reflect the amount of child signal.
Table 3: Data relating to the determination of paternity r · ^num · de,. on one. from _r ,
Con- Defi- medio no num. in . . fraction of. _í . _ SNPs no. _í „SNPs signal. _í with MCAO. _x set of SNPs parent B. set child set fi <0.05 13300 0.012 171 9 0.0007
5 ₂ fi> 0.7 3000 0.79 2370 119 0.039 [00197] Figure 1 shows the distribution of allele intensity data for AA contexts | AA and AA | BB from a sample of maternal plasma collected at 38 weeks. The B allele is measured. Note that AA | BB distribution extends significantly higher than AA | AA distribution, showing that the B allele (which is only present in the child's genome) is present in AA | BB context, but not AA | AA context. Figure 2 comes from the same sample of maternal plasma as Figure 1 and shows the distribution of the t-test statistic for the B allele, using the genotypes of 200 unrelated individuals. Two distributions are shown (the two curves): the Gaussian probability limit form and a kemel distribution. The value of tc for the biological parent is marked with a star. The p-value is less than 10-7 for the null hypothesis that the alleged father has no relationship with the child.
[00198] Table 4 shows the results of eight samples of maternal blood at different stages of pregnancy. A p-value is calculated based on the data measured from each channel (allele A and B allele) for the correct parent. If both p channel values are required to be below 0.01, then two samples are
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88/116 classified incorrectly. If only one channel is needed to pass the threshold, then all samples are correctly classified. Any number of metrics and limits can be used to confirm or exclude paternity. For example, you can use a p-value cut of 0.02, 0.005, 0.001 or 0.00001, similarly, you can require that one or both channels p-values are below a certain limit or if you could have two different limits for the different p channel values.
Table 4: p values for the two channels of eight paternity determinations.
Weeks of gestation p-value (Y) p-value (X) 11 2.3x10 ' ⁷ <10 ' ⁷ 16 0.013 <10 ' ⁷ 17 <10 ' ⁷ <10 ' ⁷ 17 <10 ' ⁷ 0.0002 20 <10 ' ⁷ <10 ' ⁷ 28 0.14 0.0048 38 <10 ' ⁷ <10 ' ⁷ 38 <10 ' ⁷ <10 ' ⁷
[00199] Table 4 shows the P values for the null hypothesis that the correct father is an independent individual. Each line corresponds to a different maternal blood sample, with the corresponding paternal genetic sample. Genetic measurements made on 200 independent males were used as controls. The curve in Figure 3 shows the distribution of the intensity rates of 200 for unrelated males and the star represents the intensity ratio for the biological father. These data could be taken from a case in which blood was collected from a mother who was 11 weeks pregnant.
[00200] Figure 4 shows the frequency of distribution (ED) curves
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89/116 cumulative for the correlation coefficient between the fetal genotypic measures and the parents' genotypic measures for three cases: (1) where both the pregnant woman and the alleged father are the biological parents of the fetus (correct, the rightmost curve) , (2) where the pregnant woman is the biological mother of the fetus, but the alleged father is not the biological father of the fetus (wrong, the middle curve) and (3) in which neither the pregnant woman nor the alleged father are the parents biological factors (wrong, left curve). The curves are cdf the correlation coefficient between the embryo's genotypic data, calculated from the data measured in a single cell and the genotypic data of the assumed parents when zero, one or two of the assumed parents are actually the genetic parents of the fetus. Note that the labels for correct and two wrong are reversed. Figure 5 shows histograms for the same three cases. Note that this histogram is made up of more than 1000 cases in which one or both parents are incorrect. The histogram of the measured rate of correlation between the fetus 'genotypic data, measured in a single cell and the parent's genotypic data assumed when zero, one or two of the assumed parents are actually the fetus' genetic parents.
[00201] Thirty-five paternity results are shown in Figure 6, using the immediate method for the paternity test. They were performed on samples collected from pregnant women with gestational ages ranging from 9 to 40 weeks. The red curve on the right represents the normalized Gaussian distribution of the statistical paternity test for 800 unrelated men. The distribution of unrelated males is different for each case, a normalized distribution is used here for visualization purposes.
[00202] The blue bars represent the normalized test statistic for the correct parent (suspect). It is evident that the correct priests are clearly separated from the independent males. Note
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90/116 that the normalized test statistic roughly approximates standard deviations, so -5 in the graph below is about 5 standard deviations from the mean. Thus, all the correct parents assumed in this cohort were confirmed as the correct parents with a significance of at least 99.9999%.
[00203] In an embodiment of the invention, knowledge of parental haplotypes can be used to increase the accuracy of the test. For example, if the two parent haplotypes are known for a given segment of a chromosome, then the knowledge that SNPs are present in cases where there is no fall out can be used to determine which SNPs should be expected for the cases in which there may be abandonment. For example, imagine a set of three SNPs that are linked, that is, they are located next to each other on the same chromosome and where the contexts of the mother and the alleged father are: AA | AB, AA | AB, AA | BA. Note that when a parent's genotype is eliminated, then AB Ψ BA, since the first of the two letters represents the alleles in the first haplotype and the second letter represents the alleles in the second haplotype. Now imagine that, for those three SNPs, a significant level of the B allele is measured for all three, in this case, the chance that the supposed parent is the correct parent is low, because the two parent haplotypes are A, A, B and B, B, A, while the fetal genotype measured is positive for B in all three SNPs, and the mother could only have contributed one A. If the father's genotype was not extinct, it would not be possible to exclude this supposed father given this set of measurements. In one embodiment, this determination of the parent haplotypes can be determined by taking measurements of diploid genomic DNA, along with measurements made of genetic haploids in one or more sperm. The use of more than one sperm can allow the determination of haplotypes more accurately, as well as the number
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91/116 reticulations may have occurred, for each of the chromosomes, together with their places, during meiosis, which formed the sperm. A method to achieve this paternal elimination can be found in more detail in the four Rabinowitz patent applications mentioned in this document.
[00204] In one modality, the determination is made exclusively of paternity using measurements from the SNP and there is no data of repetitions in simple chain is used. In one embodiment, the determination is made exclusively for paternity using both the SNP and the STR measurements. SNP data can be measured using SNP microarrays or it can be measured through sequencing. Sequencing can be irrelevant or can be targeted, for example, using circularization probes that are intended for a set of polymorphic loci or can be targeted using capture hybridization techniques. In some embodiments, genetic data can be measured by a combination of methods, for example, parental genetic data can be measured with a microarray SNP, while DNA isolated from maternal serum can be measured using target capture sequencing , where hybridization probes are used to achieve the same SNPs as are found in the SNP microarrays. In a modality a combination of the following types of data can be used to determine whether or not the alleged father is the biological father of the fetus: SNP data, STR data, passing data, microdeletion data, data insertion data , translocation or other genetic data.
[00205] In one embodiment, the method may comprise the generation of a report revealing the established paternity of the fetus or other target individual. In one embodiment, the report can be generated for the purpose of communicating the determination of paternity.
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In one embodiment, the report may include a probability that the alleged father is the biological father of the fetus. Some examples of such a report are shown inside and Figure 7 is an example of a report revealing a paternity exclusion, Figure 8 is an example of a report revealing a paternity inclusion and Figure 9 is an example of a report indicating an indeterminate result. In one embodiment, the report can comprise a graph that contains a distribution of a metric related paternity to a plurality of unrelated individuals with respect to a given fetus and mother (shown as a gray curve) and an indication of the metric for the supposed father (shown as a triangle). The distribution of unrelated males is different for each case, but, in these three reports, an effective distribution of the test statistic for the fetus and unrelated males is used here. In one embodiment, the report may also contain an indication that the alleged father is more likely to be part of the distribution of unrelated individuals (for example, Figure 7), and, therefore, the alleged father is established other than the biological father of the fetus; the fact that the triangle is in the region of the paternity exclusion chart indicates that this is a paternity exclusion. In one embodiment, the report may also contain an indication that the alleged father is more likely to be not part of the metric paternity distribution for unrelated individuals (for example, Figure 8) and the alleged father is established to be the father biological the fetus, the fact that the triangle is in the region of the inclusion graph, paternity indicates that this is an inclusion of paternity. In one embodiment, the report may also contain an indication that the measurements are indeterminate (for example, Figure 9), the fact that the triangle is in the indeterminate result region of the graph indicates that no conclusions have been made regarding
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93/116 to the establishment of the fetus' paternity.
[00206] In an embodiment of the invention, the determination of whether the alleged father is the biological father of the fetus or not is made without the use of simple series repetitions (STR). In one embodiment of the invention, the accuracy of determining paternity is increased by eliminating parental genotypes. In one embodiment of the invention, the genotypes of one or more of the parents are progressively with the use of the genetic material of one or more of the individual in relation to this parent. In one embodiment, the individual related to the father is the father parents, mother, brother, son, daughter, brother, sister, aunt, uncle, twin, clone, a gamete of the father and combinations thereof.
Another Method for Determining Paternity [00207] In one embodiment, maternal plasma and optionally other genetic material can be measured through sequencing, for example, using high-throughput sequencers, such as ILLUMINA's HISEQ or MISEQ or ο ION TORRENT da LIFE TECHNOLOGIES.
[00208] Non-invasive paternity tests can be performed on a sample of maternal blood if there is a sufficient concentration of free floating fetal DNA. In general, the fraction of fetal DNA in maternal plasma, in most cases, will be between about 2 percent to 20 percent, although it can be as low as 0.01% or as high as 40% depending, in part , gestational age. It has been shown that this fetal fraction range is sufficient for paternity testing using a single hypothesis rejection method using SNP microarrays. High throughput sequencing is by far the most accurate platform that allows mathematical modeling of the measurement response expected in each SNP for mother and child genotype combinations. In one embodiment, maternal plasma and, optionally, another
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94/116 genetic material, can be measured through sequencing, for example, using high-throughput sequencers, such as HISEQ or MISEQ from ILLUMINA or ο ION TORRENT from LIFE TECHNOLOGIES. Confidence about inclusions or exclusions of paternity can be calculated using theories of probability and / or estimation.
[00209] In one embodiment, the method for testing paternity may include the following. For a supposed father, one can calculate the probability of the sequencing data, derived from plasma, with respect to the two distinct hypotheses: (1) the alleged father is the correct (biological) father (Hc) and (2) the supposed father it is not the correct (biological) parent (Hw). The hypothesis that has the highest probability or a posteriori is then chosen. In one embodiment, this approach can be combined with a platform model which relates the plasma allele ratio to the observed number of sequenced A and B alleles. With the platform model available, it is possible to derive probabilistic odds from alleles A and B sequenced for each SNP location for each hypothesis.
[00210] One complication is that the amount of fetal fraction in maternal plasma can vary between individuals and over time. In one embodiment, the method may be responsible for this variability. There are several ways to deal with this type of variability. In one embodiment, the method can expressly estimate the fetal fraction; in another modality, the method can place a priority over the unknown quantity and integrate it over all possible values. A modality uses a prior which is like a uniform distribution from 0 to some threshold, for example, 40%. Any prior can work in theory. One method calculates probabilities of various fractions of the child, either in continuous space or over a finite division and integrates or adds up over the range,
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95/116 respectively.
[00211] Consider maternal plasma with the fetal fraction, f, and a single SNP where the expected allele ratio present in the plasma is r (based on maternal and fetal genotypes). In one embodiment, the expected allele ratio is defined as the expected fraction of A alleles in the combined maternal and fetal DNA. For the maternal genotype g _m and the genotype of the child g _c , the expected allele ratio is provided by equation 1, assuming that the genotypes are represented as allele proportions as well.
r = fg _c + (1 -f) g _m (1) [00212] The observation in the SNP comprises the number of readings mapped with each allele present, n _a and nb, which adds the reading depth d. Assuming that quality control measures were applied to the mapping probabilities, so that allele mappings and observations can be considered correct. A simple model for the observation probability is a binomial distribution which assumes that each of the readings d is extracted independently from a large pool that has an allele ratio r. Equation 2 describes this model.
P (n _a , n _b r) = p _bino (n _a ; n _a + n _b , r) = ^b ) r ⁿ ° (i - r) ⁿ > (2) [00213] When the maternal and fetal genotypes are all A or all B, the expected plasma ratio will be 0 or 1 and pbino will not be well defined. In addition, this is not desirable because unexpected alleles are sometimes observed in practice. The binomial model can be extended in a number of ways. In one embodiment, it is possible to use a corrected allele ratio ^F = 1 / (n _a + nb) to allow a small amount of unexpected allele to count. In one embodiment, it is possible to use training data to model the rate of the unexpected allele that appears over
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96/116 each SNP and use this model to correct the expected allele ratio. When the expected allele ratio is not 0 or 1, the observed allele ratio may not converge to the expected allele ratio due to amplification trends or other phenomena. The allele ratio can then be modeled as a beta distribution centered on the expected allele ratio, leading to a beta-binomial distribution for P (n _a , nb | r), which has greater variance than the binomial.
[00214] A general platform model for the response in a single SNP can be defined as F (a, b, g _c , g _m , f) (3) or the probability of observing n _a = a and nb = b based on in maternal and fetal genotypes, which also depends on the fetal fraction through equation 1.
F (a, b, g _c , g _m , f) = P (n _a = a, n _b = b | g _c , g _m , f) (3) [00215] Note that it may be possible to simplify the formula ( 3) by conditioning over a function of g _c , g _m and f, for example, using r as defined in (1) and in the binomial example in (2). The equation for F could then be written:
F (a, b, g _c , g _m , f) = P (n _a = a, n _b = b | g _c , g _m , f) = P (n _a = a, n _b = b | r ( g _c , g _m , f)) (4) [00216] In general, the functional form of F can be a binomial distribution, beta-binomial distribution, multivariate Póya distribution, an empirical distribution estimated from training data or functions similar, as discussed above. In one embodiment, the functional form of F takes different forms, depending on the hypothesis for the number of copies of the chromosome in question.
A Method For Calculating Fetal Fraction [00217] Determining the fraction of fetal DNA that is present in the mixed DNA fraction can be an integral part of a non-invasive method for determining prenatal paternity, ploidy assignment or allele assignment . In some modalities, the fetal fraction of the mixed sample can be determined based on the data
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97/116 the mother's genotypes, the father's genotypic data and the genotypic data measured from the mixed sample containing both maternal and fetal DNA. In the context of paternity testing and also, to a lesser extent, in the case of ploidy attribution, the father's identity is not known and, therefore, genotypic data of the biological father of a fetus may not be available. In these cases, it is important to have a method for determining the fetal fraction that does not require the genotype of the biological father of the fetus. Various methods are described here that allow estimating the fetal fraction. These methods are generally described, so that they are appropriate when the genotype of the biological father is available and when it is not.
[00218] For a particular chromosome, suppose we are looking at N SNPs, for which we have the following data:
• A set of NR plasma sequence measurements S = (Si, ..., Snr). In a modality, where we have (A, B) counts for alleles A and B for each SNP, s can be written as s = ((ai, bi), ..., (3n, bN)), where a, is the count on the SNP i, b, is the £ (ai + bi} = NR number b in the SNP i, and • Paternal data consisting of:
o Genotype information: mother G _m = (G _m i, ..., GmN), father Gf = (Gfi, ..., Gín), where Gmi, Ga ^and (ΑΑ, ΑΒ, BB); and / or o Sequence data measurements: NRM measurements for the parent Sm ⁼ (Sm1,, Smnr), NRF measurements for the parent Sf = (Sfi, ..., Sfnr). Similar to the above simplification, if we have (A, B) counts over each SNP Sm ^- ((am1, bml), · -, (amN, bmlSl)), Sf— ((afl, bfl), ..., ( afN, bfN)) [00219] Collectively, data from the mother, child and father can be denoted as D = (G _m , Gf, S _m , Sf, S). In one mode, data
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98/116 genotypes from both parents are available; in one modality, only genotypic data from the mother are available; in one modality, only genotypic data from the father are available; in one modality, genotypic data from none of the parents is available. In some modalities, the mathematical genotypic data can be inferred from the genotypic data measured in the mixed sample. Note that, in general, the mother's data is desirable and increases the accuracy of the algorithm, but is not necessary.
[00220] Estimated fraction of the child authentic to the fraction of the child expected based on the data:
fE (cfrlD) - Jf * P (f | D) df [00221] In one mode, you can divide the range of possible fractions of the child into a set C of finely spaced points and perform the calculations at each point which reduce the above equation for:
f = E (f | D) = ^ f * P (f | D) feC [00222] P (f | D) is probability of a fraction of the child f based on data D. One can further derive using the rule from Bayes:
PtflD) ~ P (D | f) * P (f) [00223] where P (f) is the priority weight of the particular child's fraction. In one modality, this can be derived from uninformed prior (uniform) and can be proportional to the spacing between the candidate's fractions in set C.
[00224] P (D | cfr) is the probability of certain data, based on the fraction of the child in particular, derived under the assumptions of number of copies in particular on the relevant chromosomes. In one modality, the disomy on the chromosomes used can be assumed. The probability of data on all
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SNPs are the product of data probabilities about individual SNPs.
P (DIO = i
[00225] where i denotes a given SNP, by SNP i, we have:
Σ ^p C ^D s _m, n>9cJ> H, t) * E (g _c g _m, g _.beta. H) * P (g _m | t) * P (^ | t) m> Sc [00226 ] where g _m are possible true genotypes of the mother, gt are possible true genotypes of the father, g _c are possible true genotypes of the child eg _m , gt, g _c ^fc {AA, AB, BB}.
[00227] P (g _m | i) is the general prior probability of the mother g _m genotype over SNP i, based on the population frequency known in SNP i, denoted pA ,. In particular:
píAAlpA /) = (ρ> ΐρ ³ _j = 2 (pã _; ) · (1 -, pÇBBtpAj) = (1 - pA ^ ³ [00228] Even for p (f | i), probability of the father's genotype.
[00229] Let's say it denotes the probability of obtaining the child's true genotype = c, given parents m, f and assuming hypothesis H, which we can easily calculate. For example, for a disomy:
Parenting P (c | m, f, disomy) M F AA AB BB AA AA 1 0 0 AB AA 0.5 0.5 0 BB AA 0 1 0 AA AB 0.5 0.5 0 AB AB 0.25 0.5 0.25 BB AB 0 0.5 0.5 AA BB 0 1 0 AB BB 0 0.5 0.5 BB BB 0 0 1
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100/116 [00230] Suppose ®lgm ^{p-GSP H} "Lu be likely to be kind _aa D certain data in SNP i, based on true mother genotype m, true father f genotype, genotype true son c, hypothesis H for the number of copies and fraction of the child f. It can be divided into probability of parent, child and child data as follows:
[00231] The probability of the mother's genotype data in the lllumina
Sm · in SNP i in relation to the true genotype g _m , assuming that the genotypes in lllumina are correct, it is simply:
ômi Btn [00232] In one embodiment, the probability of mother sequence data in SNP i, in the case of counts S _m i = (ami, bmi), without extra interference or trends involved, is the binomial probability defined as: P (S _m |, i) = Px | m (ami), where X | m ~ Binorn (p _m (A), ami + bm,) with Pm ^ defined as:
m AA AB BB THE B No assignment PAN) 1 0.5 0 1 0 0.5
[00233] A similar equation applies for parental probabilities.
[00234] Note that it is possible to get an answer without the parents' data, especially without the paternal data. For example, if no data paternal genotype F is available, it can be used pig br _{f fi} = i. If no paternal St sequence data is available, P (Sf | gf, i) = 1 can be used. In one embodiment, information from different chromosomes is aggregated using averages, weighted averages, or a similar function.
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Another Fetal Fraction Calculation Method [00235] Another method for determining the fetal DNA fraction in a DNA mixture is described here. In one embodiment, a version of an estimate of maximum probability of the fetal fraction f for a paternity test, ploidy test, or other purposes can be obtained without using the paternal information. Define So as the set of SNPs with the genotype of the mother 0 (AA), So, 5 as the set of SNPs with the genotype of the mother 0.5 (AB) and Si as the set of SNPs with the maternal genotype 1 (BB) . The possible fetal genotypes on So are 0 and 0.5, which results in a set of possible f_ proportions of Ro (f) = {0.2} alleles. Similarly, Ro, s = {0,5-f, 0,5,0,5 + f} f_ and Ri (f) = {1-2, 1}. All or any subset of the So, So, 5 and Si sets can be used to obtain an estimate of the child's fraction.
[00236] Define N _a o and Nbo as the vectors formed by the sequence counts for the SNPs in So, N _a o, 5 and Nbo.s similarly for So, 5 and N _a ie Nbi similarly for Si. The maximum probability estimate ide f, using all the mathema genotype sets, is defined by equation 4.
MAXF P 7 = arg (N _{a0, b0} N | f) P (N _to .5, NbO ₅ |. F) P (C i, C i _b | f) (4) [00237] Assuming that the counts of alleles for each SNP are independently conditioned on the proportion of SNPs allele in the plasma, the probabilities can be expressed as products on the SNPs in each set:
P (N _a o, Nbo | f) -P (n _as , nbs | f)
Π ³
P (N _a i, N _b i | f) = s6Si P (n _as , n _bs | f) (5) [00238] where n _as , nbs are the counts over SNPs s.
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102/116 [00239] The dependence of f is through the sets of possible proportions of Ro (f), Ro, s (f) and Ri (f) alleles. The probability of SNP P (n _as , nbs | f) can be approximated by assuming the maximum probability genotype conditioned on f. At a reasonably high fetal fraction and reading depth, the selection of the maximum probability genotype will be highly reliable. For example, at a fetal fraction of 10 percent and a reading depth of 1,000, consider a SNP where the mother has genotype 0. The expected allele ratios are 0 and 5 percent, which will be easily distinguishable at sufficiently high reading depth . Substitution of the child's genotype estimated in equation 5 results in complete equation (6) for the estimation of the fetal fraction.
f = argmaxf
Π C ^ À ^{p (} “ ^{ín6j, i))]} Π * '>.:>)) [00240] The fetal fraction must be in the range of [0.1] and, thus, the optimization can be easily implemented looking for a restricted dimension.
[00241] Another method would be the sum of the possible genotypes for each SNP that results in the expression (7) below, for P (n _a , nt> | f) for an SNP in So. The prior probability P (r) could be assumed uniform over Ro (f) or it could be based on population frequencies. The extension for groups So, 5 and Si is trivial.
P (n _a , n _b | f) = (7)
Derivation of Probabilities [00242] Confidence can be calculated from the data probabilities of the two Htf hypotheses, that is, the alleged father is the biological father and FU, that is, the alleged father, is not the biological father. The objective is to calculate P (D | H), that is, the probability of data based on the hypothesis for each hypothesis and to infer the hypothesis that is most likely.
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In one embodiment, this can be done using the Bayes rule: p (hid) ~ p (dih) * píh), where P (H) is the prior weight of the hypothesis and where P (D | H) is the probability of data based on the hypothesis.
[00243] Consider P (D | H, f), that is, the probability of data based on the hypothesis for a given fraction of the child. If a distribution on the child's fraction is available, it is possible to derive:
P (D | H) = Jp (D, f | H) df [00244] and also:
P (D | H) - Jp (D | H, f) P (flH) df [00245] Note that P (f | H) is independent of the hypothesis, that is, P (f | H) = P (f) , since the child's fraction is the same regardless of whether the alleged father is the biological father or not and any reasonable prior P (f) can be chosen, for example, a uniform prior from 0 to 50% of the fraction of child. In one mode, it is possible to use only a fraction of the child. In this case,
P (D | H) = PfDL.O [00246] Consider the probability P (D | H, f). The probability of each hypothesis is determined based on the response model, the estimated fetal fraction, the maternal genotypes, the alleged father's genotypes, the population frequencies of alleles, the plasma allele counts and SNPs. Let's say that D represents the data, as defined before.
[00247] In one modality, it is assumed that the observation of each SNP is independently conditioned on the proportion of allele in the plasma, thus, the probability of a hypothesis of paternity is the product of the probabilities of the SNPs:
P (D [H, f) = Ρ (Ο) Η, ί, ϊ)
SNPs i
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104/116 [00248] The following equations describe how one can derive the probability for a single SNP ie a single f fraction of the child. Equation 8 is a general expression for the probability of any H hypothesis which will then be divided into specific cases of Htf and Hwf. Note that the genotypes, g _m , gtf, gdf, will assume values in {AA, AB, BB} which translate into {0, 0.5, 1}, where AA = 0, AB = 0.5, BB = 1 Also, gtf denotes the genotypes of the real parent eg _df denotes the genotypes represented by the data provided to the parent. In this case, Htf, gtf and _df are equivalent.
P (DIA «, Í) = £ P (O _S „. _3áf . _Sc , f.HA) 'P (g _c 8 _m -9 ^> P (S _m i)' P (Stf i) 'P (3df i') (8) [00249] In the case of the Htf hypothesis, the alleged father is the biological father and the fetal genotypes are inherited from the mathema and genotypes of the alleged father. The above equation is simplified to:
p (d | /, w = - £ p (O s „, g„ .g _and .f _t íi = n _{t {} .i} -p {g ₍ g _n .gt _f .n = ΉΜΟ [00250 ] Still,
P (9c 9m'9tf-H = ^H tf) = PM9m'9tf) [00251] and
P (s _m | g _m , i) P (G _m | g _ni , i) P (s _f | g _tf , i) P (G _f | g _t f _/ i) P (s | g _niJ g _o f _ii) (9) [00252] for HWF, the alleged father is not the biological father. An estimate of the true paternal genotypes can be generated using the population frequencies in each SNP. Thus, the child's genotype probabilities can be determined by the mother's known genotypes and population frequencies, that is, the data do not provide additional information about the biological father's genotypes. In this case, the above equation no longer simplifies and remains as:
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P (p f, H = H _wf , l) = Σ ^p (P 9m.9df'Sc'f'H = H _W f'i) 3τη · 9 (/ · 3ά / · 3ί: ^ {0, ί), 5.1} * P (9c 9 _m '9tf'H = H _wf ) * K <J _m | 0 * P (íJt / | i) * P (9df IO [00253] Still,
P (3 _c 9m-9tf'H = H _W f) = P (âc 9m'9tf) [00254] where the only information on gtf are the priors of the population and:
^p {b 9mi 9tf>9c> f '9 ⁼ H _W f! í) ⁼
P (Smlgm, Í) P (Gmlgm <>) P (Sflgdf <íWflgdf- í) P (sIgm> gc · ^f -0 0 °) [00255] In both probability expressions, P (D | f, H, i), that is, for both hypotheses, the response model, P (s | g _m , gdf, gc, f, H) is generalized. Specific examples are mentioned elsewhere in the document in discussions about platform models in general. Some examples for the response model include binomial distribution, beta-binomial distribution, multivariate Póya distribution, an empirical distribution estimated from training data or similar functions, as discussed above.
[00256] In some modalities, the confidence C _p on the correct paternity can be calculated from the probabilities P (D | Htf) and P (D | Hwf). In one embodiment, this calculation can be done using the Bayes rule, as follows:
_{c =} ______________ ^P + P (D H _wf ) P (H _wf ) [00257] or written for a more specific case, such as a product on SNPs of the two probabilities:
, Tljjs | í / j, G _ms , + 11 $ P (n _asr ⁿ bs Gyns, G _t f, f) [00258] In another modality, confidence can be calculated as follows:
Petition 870190070429, of 7/24/2019, p. 110/134
106/116 ^p P (D | H _{t /} ) + P (D | H _{w /} ) [00259] Other reasonable probability functions are also possible.
Experimental Section
Experiment 1 [00260] Twenty-one pregnant women with confirmed paternity and gestational age between 6 and 21 weeks were enrolled. Participants voluntarily donated blood as part of our approved IRB research program and were drawn from IVF centers, OB offices and the general population in different locations across the United States. Cellless DNA (ffDNA) isolated from maternal plasma, together with DNA from the mother and the alleged father, were amplified and measured using a SNP array. A computerized method described here was used to exclude or include paternity for 21 correct parents and 36,400 incorrect parents when comparing each alleged father against a reference distribution generated from a set of more than 5,000 unrelated individuals. 20 of the 21 samples had enough fetal DNA to return results. Twenty of twenty (100%) paternity inclusions were correct. 36,382 out of 36,382 paternity exclusions were correct (100%), with 18 unassigned due to intermediate genetic similarity. There were no erroneous assignments.
[00261] The population was composed of couples who donated their blood for prenatal research. Women had to have single pregnancies, either in the first or second trimester, and confirmed paternity. Blood samples were collected from women using CELL-FREE blood tubes (STRECK) containing white blood cell preservative and genetic samples were collected from the father, either as a blood sample (EDTA) or buccal. Informed consent was obtained from all participants and the
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107/116 genetic samples were collected from patients seen in an approved IRB study.
[00262] Maternal blood was centrifuged to isolate the buffy coat and serum. Genomic DNA in the maternal and presumptive father's buffy coat and DNA in maternal serum were prepared for analysis and passed over SNP ILLUMINA INFINIUM CYTO12 arrays using standard protocols. Briefly, the DNA was isolated using the QIAGEN CIRCULATING NUCLEIC ACID kit and eluted in 45 _μ Ι of buffer according to the manufacturer's instructions. Twenty microliters of eluate were used in a blind termination reaction in 1x NEB 4 buffer, 0.42 mM dNTP and 2.5 U of T4 DNA polymerase (NEW ENGLAND BIOLABS) and incubated at 20 ° C for 30 min, then , 75 ° C for 15 min. Three microliters of binding mix (0.5 μΙ of 10x NEB 4, 1 _μ Ι of 10 mM ATP, 1 _μ Ι of PNK T4 (NEW ENGLAND BIOLABS), 0.5 _μ Ι of T4 DNA ligase (NEW ENGLAND BIOLABS))) were added and the samples incubated at 16 ° C for 24 hours, then 75 ° C for 15 min. The sample was transferred to the standard ILLUMINA INFINIUM assay together with the maternal and the alleged father's genomic DNA. In summary, 24 μΙ of DNA had the entire genome amplified at 37 ° C for 20-24 hours, followed by fragmentation and precipitation. The precipitate was then resuspended in hybridization buffer, thermally denatured and transferred to SNP Cyto12 arrangements using a TECAN EVO. The arrangements were incubated at 48 ° C for at least 16 hours, X-Stained (INFINIUM II CHEMISTRY) and washed in TECAN EVO and finally digitized. Arrangement intensities were extracted using BEADSTIDUO (ILLUMINA).
[00263] The computerized method described generated a test statistic that measures the degree of genetic similarity between the fetus and another individual. This test statistic was calculated for both the supposed
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108/116 father and for a group of more than 5,000 unrelated individuals. A single hypothesis rejection test, then, determined whether the statistic calculated for the alleged father could be excluded from the distribution formed by unrelated reference individuals. If the alleged father could be rejected from the unrelated set, then inclusion of paternity resulted; otherwise, paternity was excluded. For the 20 samples with sufficient DNA, the paternity test was performed against 20 correct parents and for 1,820 incorrect parents randomly selected.
[00264] The inclusion of paternity was attributed when the p-value of the supposed father's test statistic in the distribution of unrelated individuals was less than 10 ' ⁴ . This means that, in theory, it is expected that no more than one in 10,000 unrelated individuals will show so much genetic similarity to the fetus. An unassigned one was assigned when the p-value is between 10 ' ⁴ and 0.02. An insufficient fetal DNA assignment was made when the fetal DNA corresponded to less than 2% of the plasma DNA. The set of unrelated individuals used to generate the expected distribution was composed of individuals from a wide variety of racial origins and the determination of inclusion or exclusion of paternity was recalculated for sets of unrelated individuals of different races, including the race indicated for the supposed father. The inclusion and exclusion results were generated automatically by the algorithm and no human intervention was necessary.
[00265] In conclusion, twenty-one maternal blood samples with known paternity were tested. Twenty of the 21 samples returned results, while one had insufficient fetal DNA for analysis; this sample was taken from a woman with a gestational age of 8 weeks. Twenty of twenty (100%) results had their correct paternity confirmed, each with a p-value <10 ' ⁴ . Each
Petition 870190070429, of 7/24/2019, p. 113/134
109/116 one of 20 samples with sufficient fetal fraction was tested against a random set of 1,820 incorrect parents for a total of 36,400 individual paternity tests. 36,382 of these analyzes returned a result; 36,382 of 36,382 (100%) had their paternity correctly excluded, with a p-value of more than 10 ' ⁴ and 18 of 36,400 (0.05%) were assigned with no attribution, with a p-value of between 10' ⁴ and 0.02. There were no exclusions or incorrect paternity inclusions.
[00266] Nine of the 21 samples confirmed paternity due to fertilization control during IVF with correct paternity confirmed after fertilization through preimplantation genetic diagnosis. Twelve samples had confirmed paternity through independent paternity tests of genomic fetal / child DNA performed by the DNA Diagnostic Center, Fairfield, Ohio. Experiment 2 [00267] In one experiment, four samples of maternal plasma were prepared and amplified using a 9,600-plex hemi-grouped protocol. The samples were prepared as follows: between 15 and 40 mL of maternal blood were centrifuged to isolate the buffy coat and plasma. Maternal genomic DNA was prepared from the buffy coat and paternal DNA was prepared from a blood sample or saliva sample. Cellless DNA in maternal plasma was isolated using the QIAGEN CIRCULATING NUCLEIC ACID kit and eluted in 45 _μ Ι TE buffer according to the manufacturer's instructions. Universal binding adapters were attached to the end of each 35 μΐ molecule of purified plasma DNA and libraries were amplified for 7 cycles using adapter-specific primers. The libraries were purified with AGENCOURT AMPURE globules and eluted in 50 μΙ of water.
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110/116 [00268] 3 μΙ of DNA was amplified with 15 cycles of STA (95 ° C for 10 minutes for initial polymerase activation, followed by 15 cycles of 95 ° C for 30 s; 72 ° C for 10 s, 65 ° C for 1 min, 60 ° C for 8 min, 65 ° C for 3 minutes and 72 ° C for 30 sec and a final extension at 72 ° C for 2 min) using a 14.5 nM primer concentration of 9600 primers labeled reverses specific to the target and a direct forward specific to the library adapter at 500 nm.
[00269] The hemi-clustered PCR protocol involved a second amplification of a dilution of the products of the first STA during 15 cycles of STA (95 ° C for 10 minutes for initial polymerase activation, followed by 15 cycles of 95 ° C for 30 s, 65 ° C for 1 min; 60 ° C for 5 min, 65 ° C for 5 min and 72 ° C for 30 s and a final extension at 72 ° C for 2 min) using a reverse tag concentration of 1000 nM and a concentration of 16.6 nM for each of the 9600 target specific direct primers.
[00270] An aliquot of the STA products was then amplified by means of standard PCR for 10 cycles with 1 μΜ of barcode specific forward and reverse primers to generate barcode sequencing libraries. An aliquot from each library was mixed with different barcode libraries and purified using a centrifuge column.
[00271] In this way, 9600 initiators were used for reactions in a single well; the primers were designed to target SNPs found on chromosomes 1, 2,13,18, 21, X and Y. The amplicons were then sequenced using an ILLUMINA GAIIX sequencer. For example, approximately 3.9 million readings were generated by the sequencer, with 3.7 million readings mapping to the genome (94%) and, of these, 2.900 million readings.
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111/116 readings (74%) mapped to target SNPs with an average reading depth of 344 and a median reading depth of 255. The fetal fraction for the four samples was found to be 9.9%, 18.9 %, 16.3% and 21.2%.
[00272] Relevant maternal and paternal genomic DNA samples were amplified using a semi-grouped 9600-plex protocol and sequenced. The semi-grouped protocol is different in that it applies 9,600 external direct and reverse primers marked at 7.3 nM in the first STA. Thermocycling conditions and composition of the second STA and the barcode PCR were the same as for the hemi-grouped protocol.
[00273] The sequencing data were analyzed using the computerized methods described here and each of a set of ten unrelated men from a reference set was determined to be not the biological father of each of the unborn fetuses.
Experiment 3 [00274] In one experiment, 45 sets of cells were amplified using a semi-grouped 1200-Plex protocol, sequenced and ploidy determinations were made on three chromosomes. Note that this experiment is used to simulate the conditions for conducting paternity testing on single fetal cells obtained from maternal blood, forensic samples or where a small amount of the child's DNA is present. 15 single individual cells and 30 sets of three cells were placed in 45 individual reaction tubes for a total of 45 reactions, where each reaction contained cells from only one cell line, but the different reactions contained cells from different cell lines. The cells were prepared in 5 μΙ of wash buffer and subjected to lysis by adding 5 μΙ of lysis buffer
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112/116
ARCTURUS PICOPURE (APPLIED BIOSYSTEMS) and incubation at 56 ° C for 20 min, 95 ° C for 10 min.
[00275] DNA from single cells / three cells was amplified with 25 cycles of STA (95 ° C for 10 minutes for initial polymerase activation, then 25 cycles of 95 ° C for 30 s; '72 C for 10 s, 65 ° C for 1 min and 60 ° C for 8 min; 65 ° C for 3 min and 72 ° C for 30 sec and a final extension at 72 ° C for 2 min) using a primer concentration of 1200 labeled and forward reverse primers specific to the target.
[00276] A semi-clustered PCR protocol involved three second parallel amplification of a dilution of the first STA products for 20 cycles of STA (95 ° C for 10 minutes for initial polymerase activation, followed by 15 cycles of 95 ° C for 30 s, 65 C for '1 min, 60 ° C for 5 min, 65 ° C for 5 min and 72 ° C for 30 s and a final extension at 72 ° C for 2 min) using a specific reverse primer concentration 1000 nM target and a concentration of 60 nM for each of the 400 target specific pooled direct primers. In the three parallel 400-Plex reactions, a total of 1200 targets amplified in the first STA were thus amplified.
[00277] An aliquot of the STA products was then amplified using standard PCR for 15 cycles with 1 μΜ of target specific reverse and forward primers to generate barcode sequencing libraries. An aliquot from each library was mixed with different barcode libraries and purified using a centrifuge column.
[00278] In this way, 1200 primers were used in reactions with single cells; the primers were designed to target SNPs found on chromosomes 1, 21 and X. The amplicons
Petition 870190070429, of 7/24/2019, p. 117/134
113/116 were then sequenced using an ILLUMINA GAIIX sequencer. For example, about 3.9 million readings were generated by the sequencer, with 500,000 to 800,000 million readings mapping to the genome (74% to 94% of all readings per sample).
[00279] The relevant maternal and paternal genomic DNA samples from cell lines were analyzed using the same semi-clustered 1200-plex assay pool with a similar protocol with fewer cycles and 1200-Plex from the second sequenced STAe.
[00280] The sequencing data were analyzed using the computerized methods described here and each of a set of ten unrelated men in a reference set was determined to be not the biological father of the target individual for each of the 45 cells.
DNA of Children of Previous Pregnancy in Maternal Blood [00281] One difficulty in testing non-invasive prenatal paternity is to differentiate fetal cells from current pregnancy from fetal cells from previous pregnancies. Some believe that the genetic material from previous pregnancies is eliminated after some time, but conclusive evidence has not been shown. In one embodiment of the present description, it is possible to determine the fetal DNA present in maternal blood of paternal origin (ie, DNA that the fetus inherited from the father) using the PARENTAL SUPPORT® (PS) method and knowledge of the paternal genome. This method can use the deleted paternal genetic information. It is possible to eliminate the paternal genotype from undetected genotypic information using genetic data from grandparents (such as genetic data measured from grandfather sperm) or genetic data from other children or a sample of a miscarriage. Also, one could eliminate undeleted genetic information through HapMap-based elimination or paternal cell haplotyping. Successful haplotyping is
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114/116 demonstrated when cells stop at the mitosis stage, when chromosomes are tight bundles and using microfluidics to place separate chromosomes in separate pools. In another modality, it is possible to use the eliminated paternal haplotypic data to detect the presence of more than one parent's counterpart, which implies that the genetic material of more than one child is present in the blood. By focusing on chromosomes that are expected to be euploid in the fetus, one can rule out the possibility that the fetus will suffer from a trisomy. In addition, it is possible to determine whether the fetus' DNA is not from the current father, in which case other methods, such as the triple test, can be used to predict genetic abnormalities.
[00282] There may be other sources of fetal genetic material available via methods other than blood collection. In the case of fetal genetic material available in maternal blood, there are two main categories: (1) whole fetal cells, for example, in nucleated red blood cells or fetal erythroblasts and (2) free-floating fetal DNA. In the case of whole fetal cells, there is some evidence that fetal cells can persist in maternal blood for an extended period of time, so that it is possible to isolate a cell of a pregnant woman that contains the DNA of a child or fetus of a pregnancy previous. There is also evidence that free-floating fetal DNA is eliminated from the system in a matter of weeks. A challenge is how to determine the identity of the individual whose genetic material is contained in the cell, that is, to ensure that the measured genetic material is not from a fetus from a previous pregnancy. In one embodiment of the present description, knowledge of maternal genetic material can be used to ensure that the genetic material in question is not maternal genetic material. There are a number of methods to achieve this,
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115/116 including computerized methods, such as PARENTAL SUPPORT®, as described here or in any of the patents cited in this document.
[00283] In one embodiment of the present description, the blood collected from a pregnant woman can be separated into a fraction comprising free floating fetal DNA and a fraction comprising nucleated red blood cells. Free-floating DNA can optionally be enriched and genotypic DNA information can be measured. Based on genotypic information measured from free-floating DNA, knowledge of the maternal genotype can be used to determine aspects of the fetal genotype. These aspects may refer to ploidy status and / or a set of allele identities. Then, individual nucleated cells that are presumed or possibly of fetal origin can undergo genotyping using methods described in this document and other related patents, especially those mentioned in this document. Knowledge of the maternal genome would allow to determine whether any single blood cell is genetically maternal or not. And the aspects of the fetal genotype that were determined as described above would allow to determine whether the single blood cell is genetically derived from the fetus that is currently in gestation. In essence, this aspect of the present description makes it possible to use the mother's genetic knowledge and possibly the genetic information of other related individuals, such as the father, together with the genetic information measured from the free-floating DNA found in maternal blood to determine whether an isolated nucleated cell found in maternal blood is (a) genetically maternal, (b) genetically from the fetus currently pregnant or (c) genetically from a fetus from a previous pregnancy.
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116/116 [00284] All patents, patent applications and published references cited here are hereby incorporated by reference in their entirety. It will be appreciated that several of the features and functions described above and others or alternatives to them may desirably be combined in many other different systems or applications. Various presently unforeseen alternatives, modifications, variations or improvements can subsequently be made by those skilled in the art, which are also intended to be covered by the following claims.

权利要求:
Claims (26)
[1]
1. Ex vivo method for determining whether an alleged father is the biological father of a fetus that is unborn in a pregnant woman, characterized by the fact that it comprises:
making genotypic measurements at a plurality of polymorphic loci on the alleged parent's genetic material, wherein the plurality of polymorphic loci comprises single nucleotide polymorphisms (SNPs);
make genotypic measurements on the plurality of polymorphic loci in a mixed DNA sample from a blood sample of a pregnant woman, in which the mixed DNA sample comprises fetal DNA and maternal DNA;
determining, on a computer, the probability that the alleged father is the biological father of the fetus using the genotypic measurements made on the genetic material of the alleged father and the mixed DNA sample, and establishing whether the alleged father is the biological father of the fetus using the determined probability that the alleged father is the biological father of the fetus.
[2]
2. Method according to claim 1, characterized by the fact that it still comprises obtaining genotypic measurements in the plurality of polymorphic loci from genetic material from the mother, in which the probability that the alleged father is the biological father of the fetus is determined using genotypic measurements made on the mother's genetic material, the alleged father's genetic material and the mixed DNA sample.
[3]
3. Method according to claim 1, characterized by the fact that the mixed DNA sample comprises (i) DNA in free float from a plasma fraction of the mother's blood sample, (ii) intact maternal blood, or (iii) a fraction of blood
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2/9 maternal containing nucleated cells.
[4]
Method according to claim 3, characterized in that the fraction of nucleated cells containing maternal blood has been enriched for cells of fetal origin.
[5]
5. Method according to claim 2, characterized by the fact that the determination comprises:
calculation of a test statistic for the alleged father, in which the test statistic for the alleged father indicates a degree of genetic similarity between the alleged father and the fetus, and in which the test statistic for the alleged father is based on the genotypic measurements made on the alleged father's genetic material, the mother's genetic material, and the mixed DNA sample;
calculating a plurality of test statistics for a plurality of unrelated individuals with the fetus, where each test statistic for an unrelated individual indicates a degree of genetic similarity between the unrelated individual and the fetus, and where the test statistic for the unrelated individual is based on genotypic measurements made on the unrelated individual's genetic material, the mother's genetic material, and the mixed DNA sample;
calculating a probability that the test statistic for the alleged father is part of the distribution of the test statistic for the plurality of unrelated individuals; and determining the probability that if the alleged father is the biological father of the fetus using the probability that the test statistic for the alleged father is part of the distribution of the test statistic for the plurality of unrelated individuals.
[6]
6. Method according to claim 5, characterized by the fact that the establishment if the alleged father is the biological father of the fetus further comprises:
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3/9 establish whether the alleged father is the biological father of the fetus by rejecting the hypothesis that the alleged father is not related to the fetus, if the probability that the alleged father is the biological father of the fetus is above a maximum limit , or establish whether the alleged father is not the biological father of the fetus by not rejecting the hypothesis that the alleged father is not related to the fetus, if the probability that the alleged father is the biological father of the fetus is below a threshold minimum, or not to establish whether an alleged father is the biological father of the fetus, whether the probability is between the minimum limit and the maximum limit or whether the probability is not determined with sufficiently high confidence.
[7]
7. Method according to claim 2, characterized by the fact that determining the probability that the alleged father is the biological father of the fetus comprises:
obtaining population allele frequencies at each locus of the plurality of polymorphic loci;
creating a division of possible fetal DNA fractions in the mixed DNA sample ranging from a minimum fetal fraction limit to a maximum fetal fraction limit;
calculating a probability that the alleged father is the biological father of the fetus based on genotypic measurements made on the mother's genetic material, the alleged father's genetic material and the mixed DNA sample for each of the possible fetal fractions in the division;
determining the probability that the alleged father is the biological father of the fetus by combining the calculated probabilities that the alleged father is the biological father of the fetus for each of the possible fetal fractions in the division;
calculating a probability that the alleged father is not the biological father of the fetus based on genotypic measurements made on
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4/9 the genetic material of the mother, the genetic material of the alleged father and the mixed DNA sample, provided in the population frequencies of alleles obtained for each of the possible fetal fractions in the division; and determining the probability that the alleged father is not the biological father of the fetus by combining the calculated probabilities that the alleged father is not the biological father of the fetus for each of the possible fetal fractions in the division.
[8]
8. Method according to claim 7, characterized by the fact that calculating the probability that the alleged father is the biological father of the fetus and calculating the probability that the alleged father is not the biological father of the fetus further comprise:
calculating, for each of the plurality of polymorphic loci, the probability of sequence data observed at a particular locus using a platform response model, one or a plurality of fractions in the possible division of fetal fractions, a plurality of proportions of alleles for the mother, a plurality of allele proportions for the alleged father and a plurality of allele proportions for the fetus;
calculating a probability that the alleged father is the biological father by combining the probability of the sequence data observed at each polymorphic locus on all possible fetal fractions in the division, on the proportions of the mother's alleles in the set of polymorphic locus, on the allele proportions of the alleged father in the set of polymorphic loci and on the proportions of fetal allele in the set of polymorphic loci;
calculating a probability that the alleged father is not the biological father by combining the probability of the sequence data observed at each polymorphic locus on all possible fetal fractions in the division, on the proportions of the mother's alleles in the polymorphic locus set, about frequencies
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5/9 population for the set of polymorphic loci and on the proportions of fetal alleles in the set of polymorphic locus;
calculating a probability that the alleged father is the biological father based on the probability that the alleged father is the biological father; and calculating a probability that the alleged father is not the biological father based on the probability that the alleged father is not the biological father.
[9]
9. Method according to claim 7, characterized by the fact that the calculation of the probability that the alleged father is the biological father based on the probability that the alleged father is the biological father is performed using an estimate of maximum probability or a maximum a posteriori technique.
[10]
10. Method according to claim 7, characterized by the fact that the establishment if the alleged father is the biological father of a fetus further comprises:
establish that the alleged father is the biological father if the calculated probability that the alleged father is the biological father of the fetus is significantly greater than the calculated probability that the alleged father is not the biological father; or establish that the alleged father is not the biological father of the fetus if the calculated probability that the alleged father is not the biological father is significantly greater than the calculated probability that the alleged father is the biological father.
[11]
11. Method according to claim 7, characterized by the fact that the polymorphic loci correspond to chromosomes that have a high probability of being dysomic.
[12]
12. Method according to claim 7, characterized by the fact that the division of possible fetal DNA fractions contains only a fetal fraction and in which the fetal fraction is determined by
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6/9 using a technique selected from the group consisting of quantitative PCR, digital PCR, targeted PCR, circularization probes, other DNA amplification methods, hybridization capture probes, other preferential enrichment methods, SNP microarrays, microarrays DNA sequencing, other techniques for measuring polymorphic alleles, other techniques for measuring non-polymorphic alleles, measuring polymorphic alleles that are present in the father's genome but are not present in the mother's genome, measurement of non-polymorphic alleles that are present in the father's genome but not present in the mother's genome, measurement of alleles that are specific to the Y chromosome, comparison of the measured value of alleles inherited from the father to the measured amount of alleles inherited from the mother, maximum probability estimates, maximum techniques a posteriori and combinations thereof.
[13]
13. Method according to claim 7, characterized by the fact that the division of possible fetal fractions contains only a fetal fraction.
[14]
Method according to any one of claims 1 to 14, characterized in that the genetic material of the alleged father is obtained from a tissue selected from the group consisting of blood, somatic tissue, sperm, hair, oral sample, skin, other forensic samples and combinations thereof.
[15]
15. Method according to any one of claims 1 to 14, characterized by the fact that a confidence is calculated for the determination established whether the alleged father is the biological father of the fetus.
[16]
16. Method according to any one of claims 1 to 14, characterized in that the fraction of DNA
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7/9 fetal in the mixed DNA sample was enriched using a method selected from the group consisting of size selection, universal binding mediated by PCR, PCR with short extension times, and combinations thereof.
[17]
17. Method according to claim 2, characterized by the fact that obtaining genotypic measurements of the mother's genetic material comprises making genotypic measurements on a sample of the mother's genetic material that consists essentially of maternal genetic material.
[18]
18. Method according to claim 2, characterized by the fact that obtaining genotypic measurements of the mother's genetic material comprises:
infer which genotypic measurements from the genotypic measurements made on the mixed DNA sample are likely attributable to the mother's genetic material; and use of those genotypic measurements that were inferred to be attributable to the mother's genetic material as the genotypic measurements obtained.
[19]
19. Method according to any of claims 1 to 18, characterized by the fact that the method further comprises taking a clinical action based on the determination of established paternity, in which the clinical action is the termination of a pregnancy.
[20]
20. Method according to any one of claims 1 to 18, characterized by the fact that genotypic measurements are performed using a technique or technology selected from the group consisting of PADLOCK probes, molecular inversion circularization probes, other circularization, genotyping microarrays, SNP genotyping assays, chip-based microarrays, microarrays with globule bases,
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8/9 other SNP microarrays, other genotyping methods, Sanger DNA sequencing, pyro-sequencing, high-throughput sequencing, objective sequencing using circularization probes, objective sequencing using hybridization probe capture, reversible Dye Terminator sequencing, sequencing ligation, sequencing by hybridization, other methods of DNA sequencing, other high-performance genotyping platforms, fluorescent in situ hybridization (Fluorescent In Situ Hybridization - FISH), comparative genomic hybridization (Comparative Genomic Hybridization - CGH), arrangement CGH and multiple or combinations thereof.
[21]
21. Method according to any one of claims 1 to 19, characterized in that the performance of genotypic measurements is made on genetic material that is amplified and / or preferably enriched before being measured using a technique or technology that is selected from the group consisting of: Polymerase Chain Reaction PCR, PCR-mediated binding, PCR with degenerative oligonucleotide primer, targeted amplification, mini-PCR, universal PCR amplification, multiple displacement amplification (Multiple Displacement Amplification - MDA), allele-specific PCR, allele-specific amplification techniques, linear amplification methods, substrate DNA binding followed by another amplification method, bridge amplification, PADLOCK probes, circularization probes, capture by hybridization probes and combinations thereof .
[22]
22. Method according to any one of claims 1 to 21, characterized in that it comprises generating a report comprising the established paternity of the fetus.
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9/9
[23]
23. Report, characterized by the fact that it describes the established paternity of the fetus generated using the method as defined in any of claims 1 to 22.
[24]
24. Method according to claim 1 or 2, characterized in that the mixed DNA sample comprises free floating DNA from the mother's blood sample.
[25]
25. Method according to any one of claims 1 to 6 and 24, characterized by the fact that it further comprises estimating the fetal fraction of DNA in the mixed sample and using the estimate to establish whether the alleged father is the biological father of the fetus.
[26]
26. Method according to claim 1 or 2, characterized by the fact that it also includes the use of the fetal fraction estimate to calculate the confidence of whether the alleged father is the biological father of the fetus.

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法律状态:
2018-07-03| B06F| Objections, documents and/or translations needed after an examination request according [chapter 6.6 patent gazette]|

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2019-05-28| B06T| Formal requirements before examination [chapter 6.20 patent gazette]|

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2019-09-03| B09A| Decision: intention to grant [chapter 9.1 patent gazette]|

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2019-10-22| B16A| Patent or certificate of addition of invention granted [chapter 16.1 patent gazette]|Free format text: PRAZO DE VALIDADE: 20 (VINTE) ANOS CONTADOS A PARTIR DE 22/12/2011, OBSERVADAS AS CONDICOES LEGAIS. |

优先权:

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申请号 | 申请日 | 专利标题

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US201061426208P| true| 2010-12-22|2010-12-22|

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US201161462972P| true| 2011-02-09|2011-02-09|

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US201161448547P| true| 2011-03-02|2011-03-02|

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US201161516996P| true| 2011-04-12|2011-04-12|

---

US13/110,685|US8825412B2|2010-05-18|2011-05-18|Methods for non-invasive prenatal ploidy calling|

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US201161571248P| true| 2011-06-23|2011-06-23|

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US201161542508P| true| 2011-10-03|2011-10-03|

---

US13/300,235|US10017812B2|2010-05-18|2011-11-18|Methods for non-invasive prenatal ploidy calling|

---

PCT/US2011/066938|WO2012088456A2|2010-12-22|2011-12-22|Methods for non-invasive prenatal paternity testing|

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US13/335,043|US10113196B2|2010-05-18|2011-12-22|Prenatal paternity testing using maternal blood, free floating fetal DNA and SNP genotyping|

---