Methods for non-invasive prenatal paternity testing

Abstract

Methods for non-invasive prenatal paternity testing are disclosed herein. The method uses genetic measurements made on plasma taken from a pregnant mother, along with genetic measurements of the alleged father, and genetic measurements of the mother, to determine whether or not the alleged father is the biological father of the fetus. This is accomplished by way of an informatics based method that can compare the genetic fingerprint of the fetal DNA found in maternal plasma to the genetic fingerprint of the alleged father.

What is claimed is: 
     
         1 . A method for establishing whether an alleged father is the biological father of a fetus that is gestating in a pregnant mother, the method comprising:
 making genotypic measurements at a plurality of polymorphic loci on genetic material from the alleged father;   making genotypic measurements at the plurality of polymorphic loci on a mixed sample of DNA originating from a blood sample from the pregnant mother, wherein the mixed sample of DNA 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 from the alleged father and the mixed sample of DNA; 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 . The method of  claim 1 , further comprising obtaining genotypic measurements at the plurality of polymorphic loci from genetic material from the mother, wherein the probability that the alleged father is the biological father of the fetus is determined using the genotypic measurements made on the genetic material from the mother, the genetic material from the alleged father, and the mixed sample of DNA. 
     
     
         3 . The method of  claim 1 , wherein the polymorphic loci comprise single nucleotide polymorphisms (SNPs). 
     
     
         4 . The method of  claim 1 , wherein the mixed sample of DNA comprises (i) free floating DNA from a plasma fraction of the blood sample from the mother, (ii) maternal whole blood, or (iii) a fraction of maternal blood containing nucleated cells. 
     
     
         5 . The method of  claim 4 , wherein the fraction of maternal blood containing nucleated cells has been enriched for cells of fetal origin. 
     
     
         6 . The method of  claim 2 , wherein the determining comprises:
 calculating a test statistic for the alleged father, wherein the test statistic for the alleged father indicates a degree of genetic similarity between the alleged father and the fetus, and wherein the test statistic for the alleged father is based on the genotypic measurements made on the genetic material from the alleged father, the genetic material from the mother, and the mixed sample of DNA;   calculating a plurality of test statistics for a plurality of individuals who are genetically unrelated to the fetus, wherein each test statistic for an unrelated individual indicates a degree of genetic similarity between the unrelated individual and the fetus, and wherein the test statistic for the unrelated individual is based on genotypic measurements made on the genetic material from the unrelated individual, the genetic material from the mother, and the mixed sample of DNA;   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 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.   
     
     
         7 . The method of  claim 6 , wherein establishing whether the alleged father is the biological father of the fetus further comprises:
 establishing that the alleged father is the biological father of the fetus by rejecting a hypothesis that the alleged father is unrelated to the fetus if the probability that the alleged father is the biological father of the fetus is above an upper threshold; or   establishing that the alleged father is not the biological father of the fetus by not rejecting a hypothesis that the alleged father is unrelated to the fetus if the probability that the alleged father is the biological father of the fetus is below a lower threshold; or   not establishing whether an alleged father is the biological father of the fetus if the likelihood is between the lower threshold and the upper threshold, or if the likelihood is not determined with sufficiently high confidence.   
     
     
         8 . The method of  claim 2 , wherein the determining the probability that the alleged father is the biological father of the fetus comprises:
 obtaining population frequencies of alleles for each locus in the plurality of polymorphic loci;   creating a partition of possible fractions of fetal DNA in the mixed sample of DNA that range from a lower limit of fetal fraction to an upper limit of fetal fraction;   calculating a probability that the alleged father is the biological father of the fetus given the genotypic measurement smade on the genetic material from the mother, the genetic material from the alleged father, and the mixed sample of DNA for each of the possible fetal fractions in the partition;   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 partition;   calculating a probability that the alleged father is not the biological father of the fetus given the genotypic measurements made on the genetic material from the mother, the genetic material from the alleged father, and the mixed sample of DNA and given the obtained allele population frequencies for each of the possible fetal fractions in the partition; 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 partition,   
     
     
         9 . The method of  claim 8 , wherein 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, a likelihood of observed sequence data at a particular locus using a platform response model, one or a plurality of fractions in the possible fetal fractions partition, a plurality of allele ratios for the mother, a plurality of allele ratios for the alleged father, and a plurality of allele ratios for the fetus;   calculating a likelihood that the alleged father is the biological father by combining the likelihood of the observed sequence data at each polymorphic locus over all possible fetal fractions in the partition, over the mother allele ratios in the set of polymorphic loci, over the alleged father allele ratios in the set of polymorphic loci, and over the fetal allele ratios in the set of polymorphic loci;   calculating a likelihood that the alleged father is not the biological father by combining the likelihood of the observed sequence data at each polymorphic locus over all possible fetal fractions in the partition, over the mother allele ratios in the set of polymorphic loci, over population frequencies for the set of polymorphic loci, and over the fetal allele ratios in the set of polymorphic loci;   calculating a probability that the alleged father is the biological father based on the likelihood that the alleged father is the biological father; and   calculating a probability that the alleged father is not the biological father based on the likelihood that the alleged father is not the biological father.   
     
     
         10 . The method of  claim 8 , wherein calculating the probability that the alleged father is the biological father based on the likelihood that the alleged father is the biological father is performed using a maximum likelihood estimation, or a maximum a posteriori technique. 
     
     
         11 . The method of  claim 8 , wherein establishing whether an alleged father is the biological father of a fetus further comprises:
 establishing 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   establishing 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.   
     
     
         12 . The method of  claim 8 , wherein the polymorphic loci correspond to chromosomes that have a high likelihood of being disomic. 
     
     
         13 . The method of  claim 8 , wherein the partition of possible fractions of fetal DNA contains only one fetal fraction, and wherein the fetal fraction is determined by a technique selected from the group consisting of quantitative PCR, digital PCR, targeted PCR, circularizing probes, other methods of DNA amplification, capture by hybridization probes, other methods of preferential enrichment, 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 genome of the father but not present in the genome of the mother, measuring non-polymorphic alleles that are present in the genome of the father but not present in the genome of the mother, measuring alleles that are specific to the Y-chromosome, comparing the measured amount of paternally inherited alleles to the measured amount of maternally inherited alleles, maximum likelihood estimates, maximum a posteriori techniques, and combinations thereof. 
     
     
         14 . The method of  claim 1 , wherein the partition of possible fetal fractrions contains only one fetal fraction. 
     
     
         15 . The method of  claim 1 , wherein the alleged father's genetic material is obtained from a tissue selected from the group consisting of blood, somatic tissue, sperm, hair, buccal sample, skin, other forensic samples, and combinations thereof. 
     
     
         16 . The method of  claim 1 , wherein a confidence is computed for the established determination of whether the alleged father is the biological father of the fetus. 
     
     
         17 . The method of  claim 1 , wherein the fraction of fetal DNA in the mixed sample of DNA has been enriched using a method selected from the group consisting of size selection, universal ligation mediated PCR, PCR with short extension times, and combinations thereof. 
     
     
         18 . The method of  claim 2 , wherein obtaining genotypic measurements from the genetic material from the mother comprises making genotypic measurements on a sample of genetic material from the mother that consists essentially of maternal genetic material. 
     
     
         19 . The method of  claim 2 , wherein obtaining genotypic measurements from the genetic material from the mother comprises:
 inferring which genotypic measurements from the genotypic measurements made on the mixed sample of DNA are likely attributable to genetic material from the mother; and   using those genotypic measurements that were inferred to be attributable to genetic material from the mother as the obtained genotypic measurements.   
     
     
         20 . The method of  claim 1 , wherein the method further comprises taking a clinical action based on the established paternity determination, wherein the clinical action is termination of a pregnancy. 
     
     
         21 . The method of  claim 1 , wherein making genetotypic measurements is done by measuring genetic material using a technique or technology selected from the group consisting of padlock probes, MOLECULAR INVERSION PROBES circularizing probes, other circularizing probes, genotyping microarrays, SNP genotyping assays, chip based microarrays, bead based microarrays, other SNP microarrays, other genotyping methods, Sanger DNA sequencing, pyrosequencing, high throughput sequencing, targeted sequencing using circularizing probes, targeted sequencing using capture by hybridization probes, reversible dye terminator sequencing, sequencing by ligation, sequencing by hybridization, other methods of DNA sequencing, other high throughput genotyping platforms, fluorescent in situ hybridization (FISH), comparative genomic hybridization (CGH), array CGH, and combinations thereof. 
     
     
         22 . The method of  claim 1 , wherein making genotypic measurements is done on genetic material that is amplified and/or preferentially enriched prior to being measured using a technique or technology that is selected from the group consisting of Polymerase Chain Reaction (PCR), ligation-mediated PCR, degenerative oligonucleotide primer PCR, targeted amplification, PCR, mini-PCR, universal PCR amplification, Multiple Displacement Amplification (MDA), allele-specific PCR, allele-specific amplification techniques, linear amplification methods, ligation of substrate DNA followed by another method of amplification, bridge amplification, padlock probes, circularizing probes, capture by hybridization probes, and combinations thereof. 
     
     
         23 . The method of  claim 1 , further comprising generating a report comprising the established paternity of the fetus. 
     
     
         24 . A report disclosing the established paternity of the fetus generated using the method of  claim 1 .