Methods for non-invasive prenatal ploidy calling

Abstract

The present disclosure provides methods for determining the ploidy status of a chromosome in a gestating fetus from genotypic data measured from a mixed sample of DNA comprising DNA from both the mother of the fetus and from the fetus, and optionally from genotypic data from the mother and father. The ploidy state is determined by using a joint distribution model to create a plurality of expected allele distributions for different possible fetal ploidy states given the parental genotypic data, and comparing the expected allelic distributions to the pattern of measured allelic distributions measured in the mixed sample, and choosing the ploidy state whose expected allelic distribution pattern most closely matches the observed allelic distribution pattern. The mixed sample of DNA may be preferentially enriched at a plurality of polymorphic loci in a way that minimizes the allelic bias, for example using massively multiplexed targeted PCR.

What is claimed is: 
     
         1 . A method for measuring an amount of DNA in a biological sample, the method comprising:
 (a) performing a multiplex amplification for at least 100 polymorphic loci on one or more chromosomes expected to be disomic in a single reaction mixture, wherein the reaction mixture comprises cell-free DNA extracted from a biological sample of a subject comprising DNA of mixed origin, wherein the DNA of mixed origin comprises DNA from the subject and DNA from a genetically distinct individual, and wherein the amplification comprises ligating oligonucleotides that hybridize to target sequences comprising the polymorphic loci and amplifying the ligated oligonucleotides by PCR;   (b) measuring a quantity of each allele at a plurality of amplified polymorphic loci that comprise an allele present in the genetically distinct individual but not the subject;   (c) determining an amount of the DNA from the genetically distinct individual in the biological sample using the quantity of each allele at the polymorphic loci.   
     
     
         2 . The method of  claim 1 , further comprising determining a bias of the multiplex amplification or a bias of the microarray analysis, and using the bias to statistically correct the determined quantity of each allele at the plurality of polymorphic loci on the one or more chromosomes expected to be disomic before the quantity of each allele is used to determine the amount of the DNA from the genetically distinct individual. 
     
     
         3 . The method of  claim 1 , wherein the DNA of mixed origin comprises DNA from a transplant. 
     
     
         4 . The method of  claim 1 , wherein the polymorphic loci are SNP loci. 
     
     
         5 . The method of  claim 1 , wherein the polymorphic loci comprise more than 200 SNP loci. 
     
     
         6 . The method of  claim 1 , wherein the polymorphic loci comprise more than 500 SNP loci. 
     
     
         7 . The method of  claim 1 , wherein the polymorphic loci comprise more than 1000 SNP loci. 
     
     
         8 . The method of  claim 1 , wherein the polymorphic loci comprise more than 2000 SNP loci. 
     
     
         9 . The method of  claim 1 , wherein the polymorphic loci comprise SNP loci on chromosome 1. 
     
     
         10 . The method of  claim 1 , wherein the polymorphic loci comprise SNP loci on chromosome 2. 
     
     
         11 . The method of  claim 1 , wherein the polymorphic loci comprise SNP loci on chromosome 3. 
     
     
         12 . The method of  claim 1 , wherein the biological sample is a blood, serum, plasma, or urine sample. 
     
     
         13 . The method of  claim 1 , wherein step (b) is performed by microarray. 
     
     
         14 . The method of  claim 1 , wherein step (b) is performed by high-throughput sequencing. 
     
     
         15 . A method for measuring an amount of DNA in a biological sample, the method comprising:
 (a) performing a PCR and/or an allele-specific amplification for at least 100 SNP loci on one or more chromosomes expected to be disomic in a single reaction mixture, wherein the reaction mixture comprises cell-free DNA extracted from a biological sample of a subject comprising DNA of mixed origin, wherein the DNA of mixed origin comprises DNA from the subject and DNA from a genetically distinct individual, and wherein the amplification comprises ligating oligonucleotides that hybridize to target sequences comprising the SNP loci and amplifying the ligated oligonucleotides;   (b) measuring a quantity of each allele at a plurality of amplified SNP loci that comprise an allele present in the genetically distinct individual but not the subject;   (c) measuring an amount of the DNA from the genetically distinct individual in the biological sample using the quantity of each allele at the SNP loci and an expected quantity of each allele at the SNP loci for different DNA fractions.   
     
     
         16 . The method of  claim 15 , further comprising determining a bias of the PCR and/or allele-specific amplification or a bias of the microarray analysis, and using the bias to statistically correct the determined quantity of each allele at the plurality of SNP loci on the one or more chromosomes expected to be disomic before the quantity of each allele is used to determine the amount of the DNA from the genetically distinct individual. 
     
     
         17 . The method of  claim 15 , wherein the DNA of mixed origin comprises DNA from a transplant. 
     
     
         18 . The method of  claim 15 , wherein the biological sample is a blood, serum, plasma, or urine sample. 
     
     
         19 . The method of  claim 15 , wherein step (b) is performed by microarray. 
     
     
         20 . The method of  claim 15 , wherein step (b) is performed by high-throughput sequencing.