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 first individual in a biological sample of a second individual, comprising:
 extracting cell-free DNA of mixed origin from the biological sample, wherein the extracted cell-free DNA comprises DNA from the first individual and DNA from the second individual;   performing a multiplex targeted amplification of at least 20 polymorphic loci from the extracted cell-free DNA to obtain amplified DNA;   performing a barcoding PCR on the amplified DNA to obtain a barcoded sequencing library;   sequencing the barcoded sequencing library and measuring an amount of each allele at the polymorphic loci; and   determining the amount of the DNA from the first 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 extracted cell-free DNA comprises DNA from a fetus. 
     
     
         4 . The method of  claim 1 , wherein the extracted cell-free DNA comprises DNA from a transplant. 
     
     
         5 . The method of  claim 1 , wherein the multiplex targeted amplification is performed with a plurality of primer pairs each designed to amplify less than about 100 bp of DNA. 
     
     
         6 . The method of  claim 1 , wherein the multiplex targeted amplification is performed with a plurality of primer pairs each designed to amplify about 65-80 bp of DNA. 
     
     
         7 . The method of  claim 1 , wherein the multiplex targeted amplification comprises 10-40 PCR cycles. 
     
     
         8 . The method of  claim 1 , wherein the multiplex targeted amplification comprises amplifying 20-1,000 polymorphic loci together in the same reaction volume. 
     
     
         9 . The method of  claim 1 , wherein the multiplex targeted amplification comprises amplifying 50-500 polymorphic loci together in the same reaction volume. 
     
     
         10 . The method of  claim 1 , wherein the polymorphic loci comprise SNP loci. 
     
     
         11 . The method of  claim 1 , wherein the polymorphic loci comprise indel loci. 
     
     
         12 . The method of  claim 1 , wherein the barcoding PCR adds a sequencing tag and/or a sample index to the amplified DNA. 
     
     
         13 . The method of  claim 1 , wherein the sequencing is high-throughput sequencing. 
     
     
         14 . The method of  claim 1 , wherein the method is performed without prior knowledge of genotypes of the first and second individuals. 
     
     
         15 . The method of  claim 1 , further comprising pooling a plurality of barcoded sequencing libraries carrying different sample indexes, wherein the plurality of barcoded sequencing libraries are obtained from a plurality of samples, wherein the pool of barcoded sequencing libraries are sequenced together and for each library an amount of each allele at the polymorphic loci are measured. 
     
     
         16 . A method for preparing a preparation of amplified DNA from a biological sample of a second individual useful for measuring an amount of DNA from a first individual in the biological sample, comprising:
 (a) extracting cell-free DNA of mixed origin from the biological sample, wherein the extracted cell-free DNA comprises DNA from the first individual and DNA from the second individual;   (b) preparing a preparation of amplified DNA by: performing a multiplex targeted amplification of at least 20 polymorphic loci from the extracted cell-free DNA to obtain amplified DNA, and performing a barcoding PCR on the amplified DNA to obtain a barcoded sequencing library;   (c) analyzing the preparation of amplified DNA by: sequencing the barcoded sequencing library and measuring an amount of each allele at the polymorphic loci, thereby determining the amount of the DNA from the first individual in the biological sample.   
     
     
         17 . The method of  claim 16 , wherein the biological sample is a blood, serum, plasma, or urine sample. 
     
     
         18 . The method of  claim 16 , wherein the extracted cell-free DNA comprises DNA from a fetus. 
     
     
         19 . The method of  claim 16 , wherein the extracted cell-free DNA comprises DNA from a transplant. 
     
     
         20 . The method of  claim 16 , wherein the multiplex targeted amplification is performed with a plurality of primer pairs each designed to amplify less than about 100 bp of DNA. 
     
     
         21 . The method of  claim 16 , wherein the multiplex targeted amplification is performed with a plurality of primer pairs each designed to amplify about 65-80 bp of DNA. 
     
     
         22 . The method of  claim 16 , wherein the multiplex targeted amplification comprises 10-40 PCR cycles. 
     
     
         23 . The method of  claim 16 , wherein the multiplex targeted amplification comprises amplifying 20-1,000 polymorphic loci together in the same reaction volume. 
     
     
         24 . The method of  claim 16 , wherein the multiplex targeted amplification comprises amplifying 50-500 polymorphic loci together in the same reaction volume. 
     
     
         25 . The method of  claim 16 , wherein the polymorphic loci comprise SNP loci. 
     
     
         26 . The method of  claim 16 , wherein the polymorphic loci comprise indel loci. 
     
     
         27 . The method of  claim 16 , wherein the barcoding PCR adds a sequencing tag and/or a sample index to the amplified DNA. 
     
     
         28 . The method of  claim 16 , wherein the sequencing is high-throughput sequencing. 
     
     
         29 . The method of  claim 16 , wherein the method is performed without prior knowledge of genotypes of the first and second individuals. 
     
     
         30 . The method of  claim 16 , further comprising pooling a plurality of barcoded sequencing libraries carrying different sample indexes, wherein the plurality of barcoded sequencing libraries are obtained from a plurality of samples, wherein the pool of barcoded sequencing libraries are sequenced together and for each library an amount of each allele at the polymorphic loci are measured.