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 determining the number of copies of a chromosome or chromosome segment of interest in the genome of a gestating fetus, the method comprising:
 hybridizing DNA in a mixed sample of DNA comprising fetal DNA and maternal DNA to a microarray comprising a plurality of probes comprising a locus (i) that is known to be polymorphic and (ii) that is likely to be informative based on the rate of SNP heterozygosity in the human population for the locus;   measuring a signal from DNA in the sample binding to each of the probes in the plurality of probes on the microarray to obtain measured genetic data comprising the measured signal for each allele at the plurality of polymorphic loci on a chromosome or chromosome segment;   calculating, on a computer, allele ratios at the plurality of polymorphic loci from the measured signal for each allele at the plurality of polymorphic loci;   creating a plurality of hypotheses specifying the number of copies of the chromosome or chromosome segment of interest in genome of the fetus;   calculating, on a computer, expected allele ratios at each polymorphic locus on the chromosome or chromosome segment for each hypothesis;   calculating, on a computer, a confidence for at least one of the hypotheses using the expected allele ratios and the calculated allele ratios.   
     
     
         2 . The method of  claim 1 , wherein the sample has been isolated from maternal blood. 
     
     
         3 . The method of  claim 1 , wherein the method further comprises amplifying the DNA prior to the measuring step. 
     
     
         4 . The method of  claim 1 , wherein the method further comprises preferentially enriching the DNA at the plurality of polymorphic loci prior to the hybridization step. 
     
     
         5 . The method of  claim 4 , wherein the preferentially enriching the DNA at the plurality of polymorphic loci comprises:
 obtaining a pre-circularized probe such that the 3′ and 5′ ends are designed to hybridize to a region of DNA that is separated from the polymorphic region of the allele by a small number of bases, where the small number is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20, or a combination thereof;   hybridizing the pre-circularized probe to purified DNA from the sample;   circularizing the pre-circularized probe; and   amplifying some or all of the circularized probe.   
     
     
         6 . The method of  claim 4 , wherein the preferentially enriching the DNA at the plurality of polymorphic loci comprises:
 obtaining a forward probe such that the 3′ end of the forward probe is designed to hybridize to the region of DNA immediately upstream from the polymorphic region, and separated from the polymorphic region by a small number of bases, where the small number is selected from the group consisting of 1, 2, 3, 4, 5, 6 to 10, and 11 to 20;   obtaining a reverse probe such that the 3′ end of the reverse probe is designed to hybridize to the region of DNA immediately downstream from the polymorphic region, and separated from the polymorphic region by a small number of bases, where the small number is selected from the group consisting of 1, 2, 3, 4, 5, 6 to 10, and 11 to 20;   hybridizing the two probes to DNA in the sample of DNA; and   amplifying the DNA using the polymerase chain reaction.   
     
     
         7 . The method of  claim 4 , wherein the preferentially enriching the DNA at the plurality of polymorphic loci comprises:
 obtaining a set of hybrid capture probes;   hybridizing the hybrid capture probes to the DNA in the sample; and   physically separating the hybridized DNA from the sample of DNA from the unhybridized DNA from the sample.   
     
     
         8 . The method of  claim 7 , wherein the set of hybrid capture probes are designed to hybridize to a region that is flanking but not crossing the polymorphic allele. 
     
     
         9 . The method of  claim 8 , wherein the set of hybrid capture probes are designed to hybridize to a region that is flanking but not crossing the polymorphic allele, and wherein the length of the flanking capture probe may be selected from the group consisting of as low as about 25 bases. 
     
     
         10 . The method of  claim 4 , wherein the preferential enrichment results in an average degree of allelic bias between the sample after preferential enrichment and the sample prior to preferential enrichment of a factor of no more than a factor of 1.2. 
     
     
         11 . The method of  claim 1 , wherein the plurality of polymorphic loci comprise single nucleotide polymorphisms. 
     
     
         12 . The method of  claim 1 , wherein the step of determining the relative probability of each hypothesis uses an estimated ratio of fetal to maternal DNA in the sample. 
     
     
         13 . The method of  claim 1 , comprising using maximum likelihood estimates to select the hypothesis with the greatest probability. 
     
     
         14 . The method of  claim 1 , wherein the probabilities of each of the hypotheses are calculated using statistical techniques taken from a group consisting of a read count analysis, comparing heterozygosity rates, a statistic that is only available when parental genetic information is used, a probability of normalized genotype signals for certain parent contexts, a statistic that is calculated using an estimated fetal fraction, and combinations thereof. 
     
     
         15 . The method of  claim 1 , further comprising producing a report stating the determined confidence for at least one of the hypotheses. 
     
     
         16 . A method for determining the number of copies of a chromosome or chromosome segment of interest in the genome of a gestating fetus, the method comprising:
 obtaining a plurality primers to a locus (i) that is known to be polymorphic and (ii) that is likely to be informative based on the rate of SNP heterozygosity in the human population for the locus;   amplifying DNA in a mixed sample of DNA comprising fetal DNA and maternal DNA using the plurality of primers to obtain amplified products comprising the plurality of polymorphic loci on a chromosome or chromosome segment;   measuring an amount of the amplified products to obtain a measured quantity of each allele at the plurality of polymorphic loci on the chromosome or chromosome segment;   calculating, on a computer, allele ratios at the plurality of polymorphic loci from the measured quantity if each allele at the plurality of polymorphic loci;   creating a plurality of hypotheses specifying the number of copies of the chromosome or chromosome segment of interest in genome of the fetus;   calculating, on a computer, expected allele ratios at each polymorphic locus on the chromosome or chromosome segment for each hypothesis;   calculating, on a computer, a confidence for at least one of the hypotheses using the expected allele ratios and the calculated allele ratios.   
     
     
         17 . The method of  claim 16 , wherein the step of measuring the amount of the amplified products is performed by sequencing. 
     
     
         18 . The method of  claim 16 , wherein the method is executed for a plurality of gestating fetuses, the method further comprising:
 determining the fraction of DNA that is fetal in each of the samples; and   wherein the step of measuring the amount of the amplified products is performed by sequencing a number of amplified DNA molecules from each of the samples, where more molecules of DNA are sequenced from those samples that have a smaller fraction of fetal DNA than those samples that have a larger fraction of fetal DNA.   
     
     
         19 . The method of  claim 16 , wherein the method is executed for a plurality of gestating fetuses, and where the measuring the amount of the amplified products is performed, for each of the fetuses, by sequencing a fraction of the sample of DNA to give a first set of DNA measurements, the method further comprising:
 making a first relative probability determination for each of the hypotheses for each of the fetuses, given the first set of DNA measurements;   resequencing a second fraction of the sample from those fetuses where the first relative probability determination for each of the hypotheses indicates that a hypothesis corresponding to an aneuploid fetus has a significant probability, to give a second set of DNA measurements;   making a second relative probability determination for hypotheses for the fetuses using the second set of DNA measurements and optionally also the first set of DNA measurements; and   determining the number of copies of the chromosome or chromosome segment of interest in the genome of a fetus whose sample was resequenced by selecting the hypothesis with the greatest probability as determined by the second relative probability determination.