Methods for non-invasive prenatal ploidy calling
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.
1. A method of detecting a trisomy of at least one chromosome or chromosome segment of interest in the genome of a fetus, the method comprising:
performing a multiplex amplification to amplify at least 1,000 polymorphic loci from free floating maternal and fetal DNA in a maternal blood, plasma, or serum sample using a plurality of primers in one reaction mixture to obtain amplified products, wherein the polymorphic loci comprise single nucleotide polymorphisms (SNPs);
obtaining genetic data from amplified products;
generating a set of one or more hypotheses specifying the number of copies of one or more of the chromosomes or chromosome segments of interest present in the genome of the fetus; wherein one of the hypotheses specifies that one or more of the chromosomes or chromosome segments of interest are trisomic;
calculating expected genetic data of the fetus for each of the hypotheses;
determining the probability of the hypotheses according to a data fit between the obtained genetic data of the fetus and the expected genetic data of the fetus using a technique selected from the group consisting of maximum a- posteriori estimation and maximum likelihood estimation; wherein the probability of the hypothesis is determined without genetic data from a reference chromosome; and
outputting the hypothesis with the greatest probability as an indication that there is a trisomy in the genome of the fetus.
2. The method of claim 1 , comprising determining that more than one chromosome or chromosome segment of interest in the target individual is trisomic.
3. The method of claim 1 , wherein the target individual does not comprise a disomic chromosome.
4. The method of claim 1 , further comprising:
calculating, on a computer, allele counts at the plurality of loci from obtained genetic data;
generating, on a computer, a joint distribution model for the expected allele counts at the plurality of loci on the chromosome or chromosome segment of interest for each hypothesis;
determining, on a computer, a relative probability of each of the hypotheses using the joint distribution model and the allele counts; and
determining the number of copies of the chromosomes or chromosome segment of interest in the genome of the target individual by selecting the hypothesis with the greatest probability.
5. The method of claim 1 , wherein the method further comprises preferentially enriching the DNA in a sample at a plurality of loci prior to the step of obtaining genetic data.
6. The method of claim 5 , wherein the obtaining genetic data at the plurality of loci comprises:
obtaining a plurality of pre-circularized probes in which each probe targets one of the loci, wherein the 3′ and 5′ end of each probe is designed to hybridize to a region of DNA that is separated from the polymorphic site of the locus by a small number of bases, and wherein the small number is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21 to 25, 26 to 30, 31 to 60, or a combination thereof;
hybridizing the pre-circularized probes to DNA from the sample;
filling the gap between the hybridized probe ends using DNA polymerase;
circularizing the pre-circularized probe; and
amplifying the circularized probe.
7. The method of claim 5 , wherein the obtaining genetic data at the plurality of loci comprises:
obtaining a plurality of ligation-mediated PCR probes in which each PCR probe targets one of the loci, wherein the corresponding upstream and downstream PCR probes are designed to hybridize to a region of DNA on one strand of DNA that is separated from the polymorphic site of the locus by a small number of bases, and wherein the small number is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21 to 25, 26 to 30, 31 to 60, or a combination thereof;
hybridizing the ligation-mediated PCR probes to the DNA from the sample;
filling the gap between the ligation-mediated PCR probe ends using DNA polymerase;
ligating the ligation-mediated PCR probes; and
amplifying the ligated ligation-mediated PCR probes.
8. The method of claim 5 , wherein the obtaining genetic data at the plurality of loci comprises:
obtaining a plurality of hybrid capture probes that target the loci;
hybridizing the hybrid capture probes to the DNA in the sample; and
physically removing some or all of the unhybridized DNA from the sample of DNA.
9. The method of claim 5 , wherein the obtaining genetic data at a plurality of loci comprises:
obtaining a plurality of inner forward primers in which each primer targets one of the loci, wherein the 3′ end of each of the inner forward primers is designed to hybridize to a region of DNA upstream from the polymorphic site of the locus and separated from the polymorphic site by a small number of bases, and wherein the small number is selected from the group consisting of 1, 2, 3, 4, 5, 6 to 10, 11 to 15, 16 to 20, 21 to 25, 26 to 30, and 31 to 60 base pairs;
optionally obtaining a plurality of inner reverse primers in which each primer targets one of the loci, wherein the 3′ end of each of the inner reverse primers is designed to hybridize to a region of DNA upstream from the polymorphic site of the locus and separated from the polymorphic site by a small number of bases, and wherein the small number is selected from the group consisting of 1, 2, 3, 4, 5, 6 to 10, 11 to 15, 16 to 20, 21 to 25, 26 to 30, and 31 to 60 base pairs;
hybridizing the inner primers to the DNA; and
amplifying the DNA using the polymerase chain reaction to form amplicons.
10. The method of claim 9 , further comprising:
appending universal adapters to the DNA in the sample; and
amplifying the DNA in the sample using the polymerase chain reaction.
11. The method of claim 9 , wherein the amplifying the DNA is done in one or a plurality of individual reaction volumes, and wherein each individual reaction volume contains more than 500 different forward and reverse primer pairs.
12. The method of claim 9 , wherein the inner primers are selected by identifying primer pairs likely to form undesired primer duplexes and removing from the plurality of primers at least one of the pair of primers identified as being likely to form undesired primer duplexes.
13. The method of claim 1 , further comprising obtaining genetic data at the plurality of loci from one or both parents of the fetus.
14. The method of claim 13 , wherein the building a joint distribution model for the expected allele count probabilities of the plurality of loci on the chromosome or chromosome segment is done using the obtained genetic data from the one or both parents.
15. The method of claim 13 , wherein the obtaining genetic data from the mother is done by estimating the maternal genetic data from DNA measurements made on the sample.
16. The method of claim 1 , wherein the step of building a joint distribution model for allele count probabilities is done by using data about the probability of chromosomes crossing over at different locations in the chromosome to model dependence between polymorphic alleles on the chromosome or chromosome segment of interest.
17. The method of claim 4 , wherein the step of determining the relative probability of each hypothesis uses an estimated fraction of fetal DNA in the sample.
18. The method of claim 4 , wherein the method comprises:
combining the relative probabilities of each of the hypotheses determined using the joint distribution model and the allele count probabilities with relative probabilities of each of the hypotheses that are calculated using one or more statistical techniques selected from the group consisting of a read count analysis, comparing heterozygosity rates, a statistic that is only available when parental genetic information is used, the probability of normalized genotype signals for certain parent contexts, a statistic that is calculated using an estimated fraction of fetal DNA in the sample, and combinations thereof.
19. The method of claim 1 , wherein a confidence estimate is calculated for the trisomy determination.
20. The method of claim 1 , wherein chromosome of interest is chromosome 13, 18 or 21.
21. A method for determining whether a fetus is triploid with trisomic autosomal chromosomes, the method comprising:
performing a multiplex amplification to amplify at least 1,000 polymorphic loci from free floating maternal and fetal DNA in a material blood, plasma, or serum sample using a plurality of primers in one reaction mixture to obtain genetic data from amplified products, wherein the polymorphic loci comprise single nucleotide polymorphisms (SNPs);
obtaining genetic data from amplified products
calculating, expected genetic data of the fetus for a hypothesis in which the fetus is triploid;
calculating, expected genetic data of the fetus for at least one hypothesis in which the fetus is not triploid;
determining the probability of the hypotheses in which the fetus is triploid and the probability of the hypothesis in which the fetus is not triploid according to a data fit between the obtained genetic data of the fetus and the expected genetic data of the fetus using a technique selected from the group consisting of maximum a-posteriori estimation and maximum likelihood estimation; wherein the probability of the hypothesis is determined without genetic data from a reference chromosome; and
outputting the hypothesis with the greatest probability as an indication of whether the fetus is triploid with trisomic autosomal chromosomes.
22. The method of claim 21 , comprising:
calculating, allele counts at the plurality of loci from obtained genetic data;
generating, a joint distribution model for the expected allele counts at the plurality of loci on the chromosome or chromosome segment of interest for each hypothesis;
determining, a relative probability of each of the hypotheses using the joint distribution model and the allele counts; and
determining the number of copies of the chromosomes or chromosome segment of interest in the genome of the fetus by selecting the hypothesis with the greatest probability.
23. The method of claim 21 , wherein the step of obtaining genetic data from the fetus is performed by measuring genetic material from a sample isolated from maternal blood comprising DNA from the fetus and DNA from the mother of the fetus.
24. The method of claim 21 , further comprising obtaining genetic data at the plurality of loci from one or both parents of the fetus.
25. The method of claim 24 , wherein the building a joint distribution model for the expected allele count probabilities of the plurality of loci on the chromosome or chromosome segment is done using the obtained genetic data from the one or both parents.
26. The method of claim 23 , wherein the obtaining genetic data from the mother is done by estimating the maternal genetic data from DNA measurements made on the sample.
27. The method of claim 24 , wherein the step of building a joint distribution model for allele count probabilities is done by using data about the probability of chromosomes crossing over at different locations in the chromosome to model dependence between polymorphic alleles on the chromosome or chromosome segment of interest.
28. The method of claim 23 , wherein the step of determining the relative probability of each hypothesis uses an estimated fraction of fetal DNA in the sample.
29. The method of claim 22 , wherein the method comprises:
combining the relative probabilities of each of the hypotheses determined using the joint distribution model and the allele count probabilities with relative probabilities of each of the hypotheses that are calculated using one or more statistical techniques selected from the group consisting of a read count analysis, comparing heterozygosity rates, a statistic that is only available when parental genetic information is used, the probability of normalized genotype signals for certain parent contexts, a statistic that is calculated using an estimated fraction of fetal DNA in the sample, and combinations thereof.