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 and characterizing a preparation of amplified DNA derived from a maternal blood sample, comprising:
 (i) isolating a plurality of different free-floating DNA molecules from the maternal blood sample and tagging the free-floating DNA molecules isolated from the maternal sample with a plurality of different molecular barcodes, wherein each strand of a DNA molecule is tagged with a different molecular barcode,   (ii) performing PCR to amplify the isolated and tagged DNA,   (iii) performing hybrid capture using hybrid capture probes directed to genetic loci associated with one or more diseases or genetic states of interest to enrich the amplified DNA,   (iv) performing PCR to amplify the enriched DNA to obtain a preparation of amplified DNA,   (v) characterizing the preparation of amplified DNA by performing high-throughput DNA sequencing to generate nucleic acid sequence data for the amplified enriched DNA, and   (vi) determining from the sequence data whether the one or more fetal mutations are present in the DNA.   
     
     
         2 . A method for sequencing DNA derived from a maternal blood sample, comprising:
 (i) isolating a plurality of different free-floating DNA molecules from a maternal blood sample and tagging the free-floating DNA molecules isolated from the maternal sample with a plurality of different molecular barcodes, wherein each strand of a DNA molecule is tagged with a different molecular barcode,   (ii) performing PCR to amplify the isolated and tagged DNA molecules,   (iii) performing hybrid capture using hybrid capture probes directed to genetic loci associated with one or more diseases or genetic states of interest to enrich the amplified DNA,   (iv) performing PCR to amplify the enriched DNA, and   (v) performing high-throughput DNA sequencing to sequence the amplified enriched DNA.   
     
     
         3 . The method of  claim 1 , further comprising calculating the fetal fraction of the maternal blood sample from at least 100 SNP loci in the amplified enriched DNA. 
     
     
         4 . The method of  claim 1 , further comprising calculating the fetal fraction of the maternal blood sample from 100-1,000 SNP loci in the amplified enriched DNA. 
     
     
         5 . The method of  claim 1 , wherein the plurality of free-floating DNA molecules are tagged with up to 1,024 different molecular barcodes. 
     
     
         6 . The method of  claim 1 , wherein the plurality of free-floating DNA molecules are tagged with more than 1,024 different molecular barcodes. 
     
     
         7 . The method of  claim 2 , further comprising calculating the fetal fraction of the maternal blood sample from at least 100 SNP loci in the amplified enriched DNA. 
     
     
         8 . The method of  claim 2 , further comprising calculating the fetal fraction of the maternal blood sample from 100-1,000 SNP loci in the amplified enriched DNA. 
     
     
         9 . The method of  claim 2 , wherein the plurality of free-floating DNA molecules are tagged with up to 1,024 different molecular barcodes. 
     
     
         10 . The method of  claim 2 , wherein the plurality of free-floating DNA molecules are tagged with more than 1,024 different molecular barcodes. 
     
     
         11 . A method for preparing a preparation of amplified DNA derived from a maternal blood sample of a pregnant mother of a fetus, comprising:
 (i) isolating a plurality of different free-floating DNA molecules from the maternal blood sample, wherein the free-floating DNA molecules comprise fetal and maternal free-floating DNA, and tagging the free-floating DNA molecules isolated from the maternal sample with a plurality of different molecular barcodes   (ii) performing PCR to amplify the isolated and tagged DNA,   (iii) performing hybrid capture using hybrid capture probes directed to single nucleotide polymorphism (SNP) target loci of interest to enrich the amplified DNA,   (iv) performing PCR to amplify the enriched DNA to obtain a preparation of amplified DNA,   (v) characterizing the preparation of amplified DNA by performing high-throughput DNA sequencing to obtain amounts of SNP target loci based on SNP allele ratios, without prior knowledge of parental genotypes, and determining the fraction of fetal cell-free DNA in the maternal blood sample using the amounts of SNP target loci.   
     
     
         12 . The method of  claim 11 , wherein the fetal fraction of the maternal blood sample is determined from at least 100 SNP loci in the preparation of amplified DNA. 
     
     
         13 . The method of  claim 11 , wherein the fetal fraction of the maternal blood sample is determined from 100-1,000 SNP loci in the preparation of amplified DNA. 
     
     
         14 . The method of  claim 11 , wherein the plurality of free-floating DNA molecules are tagged with up to 1,024 different molecular barcodes. 
     
     
         15 . The method of  claim 11 , wherein the plurality of free-floating DNA molecules are tagged with more than 1,024 different molecular barcodes.