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 sample of DNA from the mother of the fetus and from the fetus, and from genotypic data from the mother and optionally also from the father. The ploidy state is determined by using a joint distribution model to create a set 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. In an embodiment, the mixed sample of DNA may be preferentially enriched at a plurality of polymorphic loci in a way that minimizes the allelic bias.

What is claimed is: 
     
         1 . A method for preparing a preparation of amplified and enriched DNA derived from a biological sample comprising cell-free DNA of maternal origin and cell-free DNA of fetal origin useful for fetal fraction analysis, 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 and enriched DNA by: ligating adaptors comprising at least one universal amplification sequence to the extracted DNA to obtain adaptor-ligated DNA, amplifying the adaptor-ligated DNA using at least one universal primer to obtain amplified DNA, performing targeted enrichment of 100 to 20,000 polymorphic loci from the amplified DNA; and   (c) analyzing the amplified and enriched DNA by: performing next-generation sequencing to obtain quantitative measurements of different alleles at each polymorphic locus, and estimating the fetal fraction of the cell-free DNA in the biological sample using the quantitative measurements.   
     
     
         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 targeted enrichment comprises performing targeted enrichment of 1,000 to 10,000 polymorphic loci from the amplified DNA. 
     
     
         4 . The method of  claim 1 , wherein the polymorphic loci are SNP loci. 
     
     
         5 . The method of  claim 1 , wherein the targeted enrichment comprises performing targeted multiplex PCR to amplify the polymorphic loci in one reason mixture. 
     
     
         6 . The method of  claim 1 , wherein the targeted enrichment comprises enriching the polymorphic loci using hybrid capture probes. 
     
     
         7 . The method of  claim 1 , further comprising the step of incorporating sequencing-compatible adapters to the amplified and enriched DNA. 
     
     
         8 . The method of  claim 1 , further comprising determining an allelic bias, wherein the allelic bias is the average difference between an allelic ratio at the polymorphic loci in the amplified DNA after enrichment and the corresponding allelic ratio at the polymorphic loci in the amplified DNA before enrichment, wherein the allelic ratio is the measured quantity of the second most frequent allele at a polymorphic locus divided by the total measured quantity of all alleles at the polymorphic locus, and using the allelic bias to correct the quantitative measurements of different alleles at the polymorphic locus and the estimated fetal fraction. 
     
     
         9 . The method of  claim 8 , wherein the average difference between the allelic ratio at the polymorphic loci in the amplified DNA after enrichment and the corresponding allelic ratio at the polymorphic loci in the amplified DNA before enrichment is no more than a factor of 2. 
     
     
         10 . The method of  claim 8 , wherein the average difference between the allelic ratio at the polymorphic loci in the amplified DNA after enrichment and the corresponding allelic ratio at the polymorphic loci in the amplified DNA before enrichment is no more than a factor of 1.1. 
     
     
         11 . A method for preparing a preparation of amplified and enriched DNA derived from a maternal blood, plasma, or serum sample comprising cell-free DNA of maternal origin and cell-free DNA of fetal origin useful for fetal fraction analysis, comprising:
 (a) extracting cell-free DNA from the 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 preparation of amplified and enriched DNA by: ligating adaptors comprising at least one universal amplification sequence to the extracted DNA to obtain adaptor-ligated DNA, amplifying the adaptor-ligated DNA using at least one universal primer to obtain amplified DNA, performing targeted enrichment of 100 to 20,000 SNP loci from the amplified DNA; and   (c) analyzing the amplified and enriched DNA by: performing next-generation sequencing to obtain quantitative measurements of different alleles at each SNP locus, and estimating the fetal fraction of the cell-free DNA in the maternal blood, plasma, or serum sample using the quantitative measurements.   
     
     
         12 . The method of  claim 11 , wherein the targeted enrichment comprises performing targeted enrichment of 1,000 to 10,000 SNP loci from the amplified DNA. 
     
     
         13 . The method of  claim 11 , wherein the targeted enrichment comprises performing targeted multiplex PCR to amplify the SNP loci in one reason mixture. 
     
     
         14 . The method of  claim 11 , wherein the targeted enrichment comprises enriching the SNP loci using hybrid capture probes. 
     
     
         15 . The method of  claim 11 , further comprising the step of incorporating sequencing-compatible adapters to the amplified and enriched DNA. 
     
     
         16 . The method of  claim 11 , further comprising determining an allelic bias, wherein the allelic bias is the average difference between an allelic ratio at the SNP loci in the amplified DNA after enrichment and the corresponding allelic ratio at the SNP loci in the amplified DNA before enrichment, wherein the allelic ratio is the measured quantity of the second most frequent allele at a SNP locus divided by the total measured quantity of all alleles at the SNP locus, and using the allelic bias to correct the quantitative measurements of different alleles at the SNP locus and the estimated fetal fraction. 
     
     
         17 . The method of  claim 16 , wherein the average difference between the allelic ratio at the SNP loci in the amplified DNA after enrichment and the corresponding allelic ratio at the SNP loci in the amplified DNA before enrichment is no more than a factor of 2. 
     
     
         18 . The method of  claim 16 , wherein the average difference between the allelic ratio at the SNP loci in the amplified DNA after enrichment and the corresponding allelic ratio at the SNP loci in the amplified DNA before enrichment is no more than a factor of 1.1.