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 preparing a preparation of amplified DNA derived from a biological sample of a mother of a fetus useful for determining a ploidy state of a target chromosome of the fetus, comprising:
 (a) extracting cell-free DNA from the biological sample, wherein the extracted DNA comprises a mixture of cell-free DNA of maternal origin and cell-free DNA of fetal origin;   (b) preparing a preparation of amplified DNA by: performing multiplex PCR on the extract DNA to amplify 100 to 20,000 polymorphic loci in one reaction mixture, and performing universal PCR to amplify products of the multiplex PCR to obtain a preparation of amplified DNA, wherein a sample barcode and a sequencing-compatible adaptor are incorporated into the amplified DNA;   (c) analyzing the preparation of amplified DNA by performing next-generation sequencing on the amplified DNA to obtain quantitative measurements of different alleles at the polymorphic loci, and determining a ploidy state of the target chromosome using the quantitative measurements of different alleles at the polymorphic loci.   
     
     
         2 . The method of  claim 1 , wherein the biological sample is a maternal blood, plasma, or serum sample. 
     
     
         3 . The method of  claim 1 , wherein the multiplex PCR comprises amplifying 1,000 to 10,000 polymorphic loci in one reaction mixture. 
     
     
         4 . The method of  claim 1 , wherein the multiplex PCR comprises amplifying 2,000 to 5,000 polymorphic loci in one reaction mixture. 
     
     
         5 . The method of  claim 1 , wherein the polymorphic loci are SNP loci. 
     
     
         6 . The method of  claim 1 , further comprising estimating the fetal fraction of the cell-free DNA in the biological sample using the quantitative measurements of different alleles at the polymorphic loci. 
     
     
         7 . The method of  claim 1 , wherein a plurality of the polymorphic loci map to chromosomes 13, 18, and/or 21 and the method comprises determining a ploidy state of chromosomes 13, 18, and/or 21 using the quantitative measurements of different alleles at the polymorphic loci. 
     
     
         8 . The method of  claim 1 , wherein a plurality of the polymorphic loci map to a reference chromosome expected to be disomic. 
     
     
         9 . The method of  claim 1 , wherein a Z-score is determined using the quantitative measurements of different alleles at the polymorphic loci on the target chromosome and a reference chromosome, and the ploidy state of the target chromosome is determined using the Z-score. 
     
     
         10 . The method of  claim 1 , wherein the amplified DNA from multiple samples are pooled and sequenced in a single sequencing lane. 
     
     
         11 . A method for preparing a preparation of amplified DNA derived from a maternal blood, plasma, or serum sample of a mother of a fetus useful for determining a ploidy state of a target chromosome of the fetus, comprising:
 (a) extracting cell-free DNA from the biological sample, wherein the extracted DNA comprises a mixture of cell-free DNA of maternal origin and cell-free DNA of fetal origin;   (b) preparing a preparation of amplified DNA by: performing multiplex PCR on the extract DNA to amplify 100 to 20,000 SNP loci in one reaction mixture, and performing universal PCR to amplify products of the multiplex PCR to obtain a preparation of amplified DNA, wherein a sample barcode and a sequencing-compatible adaptor are incorporated into the amplified DNA;   (c) analyzing the preparation of amplified DNA by performing next-generation sequencing on the amplified DNA to obtain quantitative measurements of different alleles at the SNP loci, and determining a ploidy state of the target chromosome using the quantitative measurements of different alleles at the SNP loci.   
     
     
         12 . The method of  claim 11 , wherein the multiplex PCR comprises amplifying 1,000 to 10,000 SNP loci in one reaction mixture. 
     
     
         13 . The method of  claim 11 , wherein the multiplex PCR comprises amplifying 2,000 to 5,000 SNP loci in one reaction mixture. 
     
     
         14 . The method of  claim 11 , further comprising estimating the fetal fraction of the cell-free DNA in the biological sample using the quantitative measurements of different alleles at the SNP loci. 
     
     
         15 . The method of  claim 11 , wherein a plurality of the SNP loci map to chromosomes 13, 18, and/or 21 and the method comprises determining a ploidy state of chromosomes 13, 18, and/or 21 using the quantitative measurements of different alleles at the SNP loci. 
     
     
         16 . The method of  claim 11 , wherein a plurality of the SNP loci map to a reference chromosome expected to be disomic. 
     
     
         17 . The method of  claim 11 , wherein a Z-score is determined using the quantitative measurements of different alleles at the SNP loci on the target chromosome and a reference chromosome, and the ploidy state of the target chromosome is determined using the Z-score. 
     
     
         18 . The method of  claim 11 , wherein the amplified DNA from multiple samples are pooled and sequenced in a single sequencing lane.