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 non-naturally occurring composition of amplified DNA derived from a biological sample comprising cell-free DNA of maternal origin and cell-free DNA of fetal origin useful for estimating fetal fraction, 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 composition of amplified DNA by: performing multiplex PCR on the extract DNA to amplify 50 to 20,000 loci together in the same reaction mixture, and performing universal PCR to amplify products of the multiplex PCR to obtain a non-naturally occurring composition of amplified DNA, wherein a sequencing adaptor is incorporated into the amplified DNA, and wherein the amplification products may be derived from cell-free DNA of fetal origin and cell-free DNA of maternal origin.   
     
     
         2 . The method of  claim 1 , further comprising analyzing the non-naturally occurring composition of amplified DNA by: performing next-generation sequencing on the amplified DNA to obtain quantitative measurements of different alleles at the loci, and estimating the fetal fraction of the cell-free DNA in the biological sample using the quantitative measurements of different alleles at some or all of the loci. 
     
     
         3 . The method of  claim 1 , wherein the biological sample is a maternal blood, plasma, or serum sample. 
     
     
         4 . The method of  claim 1 , wherein the multiplex PCR comprises amplifying 50 to 2,000 loci together in the same reaction mixture. 
     
     
         5 . The method of  claim 1 , wherein the multiplex PCR comprises amplifying 50 to 500 loci together in the same reaction mixture. 
     
     
         6 . The method of  claim 1 , wherein the loci comprise single nucleotide polymorphism or variant loci. 
     
     
         7 . The method of  claim 1 , wherein a plurality of the 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 loci. 
     
     
         8 . The method of  claim 1 , wherein a plurality of the loci map to a reference chromosome expected to be disomic. 
     
     
         9 . The method of  claim 1 , wherein the amplified DNA from multiple samples are pooled and sequenced in a single sequencing lane. 
     
     
         10 . A method for preparing a non-naturally occurring composition of amplified DNA derived from a biological sample comprising cell-free DNA of maternal origin and cell-free DNA of fetal origin useful for estimating fetal fraction, comprising:
 (a) extracting cell-free DNA from the biological sample, wherein the biological sample is a maternal blood, plasma, or serum 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 non-naturally occurring composition of amplified DNA by: performing multiplex PCR on the extract DNA to amplify 50 to 20,000 SNP loci together in the same reaction mixture, and performing universal PCR to amplify products of the multiplex PCR to obtain a non-naturally occurring composition of amplified DNA, wherein a sample index and a sequencing adaptor are incorporated into the amplified DNA.   
     
     
         11 . The method of  claim 10 , further comprising analyzing the non-naturally occurring composition of amplified DNA by: performing next-generation sequencing on the amplified DNA to obtain quantitative measurements of different alleles at the SNP loci, and estimating the fetal fraction of the cell-free DNA in the biological sample using the quantitative measurements of different alleles at some or all of the SNP loci. 
     
     
         12 . The method of  claim 10 , wherein the multiplex PCR comprises amplifying 50 to 2,000 SNP loci together in the same reaction mixture. 
     
     
         13 . The method of  claim 10 , wherein the multiplex PCR comprises amplifying 50 to 500 SNP loci together in the same reaction mixture. 
     
     
         14 . The method of  claim 10 , 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. 
     
     
         15 . The method of  claim 10 , wherein a plurality of the SNP loci map to a reference chromosome expected to be disomic. 
     
     
         16 . The method of  claim 10 , wherein the amplified DNA from multiple samples are pooled and sequenced in a single sequencing lane.