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 deoxyribonucleic acid (DNA) fraction from a biological sample useful for analyzing genotypes for DNA from cancer cells in a biological sample, comprising:
 (a) extracting cell-free DNA from the biological sample, wherein the extracted cell-free DNA comprises DNA from cancer cells;   (b) producing a fraction of the DNA extracted in (a) by performing targeted multiplex amplification on the cell-free DNA extracted in (a) to amplify at least 10 different target loci in one reaction volume, followed by a nested PCR amplification to amplify the at least 10 different target loci in one reaction volume;   (c) analyzing the fraction of the DNA produced in (b) by performing high-throughput sequencing on the amplified DNA to obtain sequence reads and determining the sequence of the target loci based on the sequence reads.   
     
     
         2 . The method of  claim 1 , wherein the biological sample is a blood, plasma, serum, or urine sample. 
     
     
         3 . The method of  claim 1 , wherein the target loci comprise single nucleotide polymorphism or variant loci. 
     
     
         4 . The method of  claim 1 , wherein the targeted multiplex amplification simultaneously amplifies at least 20 different target loci in one reaction volume. 
     
     
         5 . The method of  claim 1 , wherein the targeted multiplex amplification simultaneously amplifies at least 100 different target loci in one reaction volume. 
     
     
         6 . The method of  claim 1 , wherein the nested PCR amplification simultaneously amplifies at least 20 different target loci in one reaction volume. 
     
     
         7 . The method of  claim 1 , wherein the nested PCR amplification simultaneously amplifies at least 100 different target loci in one reaction volume. 
     
     
         8 . The method of  claim 1 , wherein the nested PCR amplification is semi-nested PCR. 
     
     
         9 . The method of  claim 1 , wherein the nested PCR amplification is hemi-nested PCR. 
     
     
         10 . The method of  claim 1 , wherein the nested PCR amplification is one-sided nested PCR. 
     
     
         11 . The method of  claim 1 , wherein the nested PCR amplification is fully nested PCR. 
     
     
         12 . The method of  claim 1 , wherein the amplified DNA are tagged with molecular barcodes. 
     
     
         13 . The method of  claim 1 , wherein at least 80% of the sequence reads map to the target loci. 
     
     
         14 . The method of  claim 1 , wherein at least 90% of the sequence reads map to the target loci. 
     
     
         15 . The method of  claim 1 , wherein the method further comprises barcoding PCR to introduce a sample barcode and a sequencing tag, and wherein amplified DNA from multiple samples are pooled together and sequenced in a single sequencing lane. 
     
     
         16 . The method of  claim 1 , wherein the method further comprises determining whether the target loci comprise one or more mutations associated with cancer. 
     
     
         17 . The method of  claim 1 , wherein the biological sample is a blood, plasma, serum, or urine sample, wherein the targeted multiplex amplification simultaneously amplifies at least 20 different single nucleotide polymorphism or variant loci in one reaction volume, and wherein the nested PCR amplification simultaneously amplifies at least 20 different single nucleotide polymorphism or variant loci in one reaction volume. 
     
     
         18 . The method of  claim 1 , wherein the biological sample is a blood, plasma, serum, or urine sample, wherein the targeted multiplex amplification simultaneously amplifies at least 100 different single nucleotide polymorphism or variant loci in one reaction volume, and wherein the nested PCR amplification simultaneously amplifies at least 100 different single nucleotide polymorphism or variant loci in one reaction volume. 
     
     
         19 . The method of  claim 17 , wherein at least 80% of the sequence reads map to the target loci, and wherein the method further comprises determining whether the target loci comprise one or more mutations associated with cancer. 
     
     
         20 . The method of  claim 18 , wherein at least 90% of the sequence reads map to the target loci, and wherein the method further comprises determining whether the target loci comprise one or more mutations associated with cancer.