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 amplifying target loci from DNA of mixed origin, wherein the target loci comprise more than 100 polymorphic loci, the method comprising:
 a) extracting cell-free DNA of mixed origin from a biological sample of a subject, wherein cell-free DNA comprises DNA from the subject and DNA from a genetically distinct individual;   b) enriching the extracted cell-free DNA by:
 i) for each of the target loci, hybridizing an upstream and a downstream ligation-mediated PCR probes to a region of the DNA that comprises the target locus; 
 ii) ligating the PCR probes that are hybridized to the same region of the DNA; and 
 iii) amplifying the ligated PCR probes by PCR, thereby amplifying the target loci, wherein the more than 100 polymorphic loci are amplified in a single reaction mixture; and 
   c) determining the sequences of the amplified target loci.   
     
     
         2 . The method of  claim 1 , wherein the DNA of mixed origin comprises DNA from a transplant. 
     
     
         3 . The method of  claim 1 , wherein the polymorphic loci are SNP loci. 
     
     
         4 . The method of  claim 1 , wherein the target loci comprise more than 200 SNP loci. 
     
     
         5 . The method of  claim 1 , wherein the target loci comprise more than 500 SNP loci. 
     
     
         6 . The method of  claim 1 , wherein the target loci comprise more than 1000 SNP loci. 
     
     
         7 . The method of  claim 1 , wherein the target loci comprise more than 2000 SNP loci. 
     
     
         8 . The method of  claim 1 , wherein the target loci comprise SNP loci on chromosome 1. 
     
     
         9 . The method of  claim 1 , wherein the target loci comprise SNP loci on chromosome 2. 
     
     
         10 . The method of  claim 1 , wherein the target loci comprise SNP loci on chromosome 3. 
     
     
         11 . The method of  claim 1 , wherein for each of the target loci, each of the upstream and the downstream ligation-mediated PCR probe hybridizes to the cell-free DNA within a region of DNA that is separated by 60 nucleotides or less from the target locus. 
     
     
         12 . The method of  claim 1 , wherein the method further comprises determine the genotypes at the polymorphic loci and measuring an amount of one or more alleles at the polymorphic loci. 
     
     
         13 . The method of  claim 1 , wherein the method further comprises measuring an amount of the DNA from the genetically distinct individual present in the biological sample. 
     
     
         14 . The method of  claim 1 , wherein the biological sample is a blood, serum, plasma, or urine sample. 
     
     
         15 . The method of  claim 1 , wherein the sequences of the amplified target loci are determined by microarray. 
     
     
         16 . The method of  claim 1 , wherein the sequences of the amplified target loci are determined by high-throughput sequencing. 
     
     
         17 . A method for measuring target loci from DNA of mixed origin, wherein the target loci comprise more than 100 SNP loci, the method comprising:
 a) extracting cell-free DNA of mixed origin from a biological sample of a subject, wherein cell-free DNA comprises DNA from the subject and DNA from a transplant;   b) enriching the extracted cell-free DNA by:
 i) for each of the target loci, hybridizing an upstream and a downstream ligation-mediated PCR probes to a region of the DNA that comprises the target locus; 
 ii) ligating the PCR probes that are hybridized to the same region of the DNA; and 
 iii) amplifying the ligated PCR probes by PCR, thereby amplifying the target loci, wherein the more than 100 SNP loci are amplified in a single reaction mixture; and 
   c) determine the genotypes at the SNP loci and measuring an amount of one or more alleles at the SNP loci, and   d) measuring an amount of the DNA from the transplant present in the biological sample using the amount of one or more alleles at the SNP loci.   
     
     
         18 . The method of  claim 17 , wherein the biological sample is a blood, serum, plasma, or urine sample. 
     
     
         19 . The method of  claim 17 , wherein step (c) is performed by microarray. 
     
     
         20 . The method of  claim 17 , wherein step (c) is performed by high-throughput sequencing.