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 from a genetically distinct individual in a biological sample, comprising:
 extracting cell-free DNA of mixed origin from a biological sample of a subject, wherein the cell-free DNA comprises DNA from the subject and DNA from a genetically distinct individual;   pre-amplifying more than 100 target loci from the cell-free DNA using more than 100 primer pairs in a single reaction volume to obtain pre-amplified DNA;   amplifying a plurality of subpools of the target loci in parallel, each from one of multiple aliquots of the pre-amplified DNA, to obtain amplified DNA;   sequencing the amplified DNA and measuring an amount of one or more alleles at the target loci; and   measuring an amount of DNA from the genetically distinct individual in the biological sample.   
     
     
         2 . The method of  claim 1 , wherein the biological sample is a blood, serum, plasma, or urine sample. 
     
     
         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 DNA of mixed origin comprises DNA from a fetus. 
     
     
         5 . The method of  claim 1 , wherein the method comprises dividing the pre-amplified DNA into multiple aliquots, amplifying the plurality of subpools of the target loci in parallel in individual reaction volumes, and pooling the amplified DNA from the individual reaction volumes. 
     
     
         6 . The method of  claim 5 , wherein the method further comprises performing a barcoding PCR to add a sequencing adaptor and a sample index to the pooled amplified DNA. 
     
     
         7 . The method of  claim 6 , wherein the method comprises sequencing the barcoded amplified DNA obtained from multiple samples together in a single sequencing lane. 
     
     
         8 . The method of  claim 1 , wherein the pre-amplifying step comprises 10-30 PCR cycles. 
     
     
         9 . The method of  claim 1 , wherein 50-5,000 target loci are pre-amplified in a single reaction volume. 
     
     
         10 . The method of  claim 1 , wherein 50-500 target loci are pre-amplified in a single reaction volume. 
     
     
         11 . The method of  claim 1 , wherein more than 200 target loci are pre-amplified in a single reaction volume. 
     
     
         12 . The method of  claim 1 , wherein more than 500 target loci are pre-amplified in a single reaction volume. 
     
     
         13 . The method of  claim 1 , wherein the target loci are SNP loci. 
     
     
         14 . The method of  claim 1 , wherein the target loci comprise SNP loci on chromosome 1. 
     
     
         15 . The method of  claim 1 , wherein the target loci comprise SNP loci on chromosome 2. 
     
     
         16 . The method of  claim 1 , wherein the target loci comprise SNP loci on chromosome 3. 
     
     
         17 . The method of  claim 1 , wherein the sequencing is high-throughput sequencing. 
     
     
         18 . The method of  claim 1 , wherein the method is performed without prior knowledge of genotypes of the genetically distinct individual. 
     
     
         19 . A method for measuring an amount of DNA from a genetically distinct individual in a biological sample, comprising:
 extracting cell-free DNA of mixed origin from a biological sample of a subject, wherein the cell-free DNA comprises DNA from the subject and DNA from a genetically distinct individual, wherein the biological sample is a blood, serum, plasma, or urine sample;   pre-amplifying more than 100 SNP loci on one or more chromosomes expected to be disomic from the cell-free DNA using more than 100 primer pairs in a single reaction volume to obtain pre-amplified DNA;   dividing the pre-amplified DNA into multiple aliquots; amplifying a plurality of subpools of the SNP loci in parallel in individual reaction volumes, each from one of the multiple aliquots of the pre-amplified DNA, to obtain amplified DNA; and pooling the amplified DNA from the individual reaction volumes;   sequencing the amplified DNA and measuring an amount of each allele at a plurality of amplified SNP loci that comprise an allele present in the genetically distinct individual but not the subject; and   measuring an amount of DNA from the genetically distinct individual in the biological sample.   
     
     
         20 . A method for measuring an amount of DNA from a genetically distinct individual in a biological sample, comprising:
 extracting cell-free DNA of mixed origin from a biological sample of a subject, wherein the cell-free DNA comprises DNA from the subject and DNA from a genetically distinct individual, wherein the biological sample is a blood, serum, plasma, or urine sample;   pre-amplifying more than 100 SNP loci on one or more chromosomes expected to be disomic from the cell-free DNA using more than 100 primer pairs in a single reaction volume to obtain pre-amplified DNA;   dividing the pre-amplified DNA into multiple aliquots; amplifying a plurality of subpools of the SNP loci in parallel in individual reaction volumes, each from one of the multiple aliquots of the pre-amplified DNA, to obtain amplified DNA; and pooling the amplified DNA from the individual reaction volumes;   performing a barcoding PCR to add a sequencing adaptor and a sample index to the amplified DNA;   sequencing the amplified DNA obtained from multiple samples together in a single sequencing lane, and for each sample measuring an amount of each allele at a plurality of amplified SNP loci that comprise an allele present in the genetically distinct individual but not the subject; and   measuring an amount of DNA from the genetically distinct individual in the biological sample.