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 for preparing and characterizing a preparation of amplified DNA derived from a maternal blood sample of a pregnant mother of a fetus, comprising:
a) isolating DNA from the maternal blood sample, wherein the DNA comprises fetal and maternal cell-free DNA;
b) performing multiplex PCR on the cell-free DNA to amplify at least 100 single nucleotide polymorphism (SNP) target loci in one reaction mixture, wherein the multiplex PCR is performed with primers designed to reduce primer dimer formation, and further performing universal PCR to amplify products of the multiplex PCR to obtain a preparation of amplified DNA, wherein a sample barcode and a sequencing-compatible adaptor are incorporated into the amplified DNA during the multiplex PCR or the universal PCR;
c) characterizing the preparation of amplified DNA by performing next-generation sequencing on the amplified DNA of step b) to obtain amounts of the 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 the target loci.
2. The method of claim 1 , wherein the method further comprises determining a ploidy state of a target chromosome of the fetus using the measured amounts of at least two of the target loci.
3. The method of claim 2 , wherein one or more of the target loci map to chromosomes X, Y, 13, 18, and/or 21 and the method further comprises determining a ploidy state for chromosomes X, Y, 13, 18, and/or 21 using the measured amounts of the target loci.
4. The method of claim 3 , wherein one or more of the target loci are from a reference chromosome.
5. The method of claim 2 , wherein measuring the amounts of target loci comprises counting the number of reads that map to a given chromosome.
6. The method of claim 2 , wherein the measuring provides bulk genotypic measurements of at least some of the target loci, and wherein the bulk genotypic measurements are used to determine the ploidy state of the target chromosome.
7. The method of claim 2 , wherein the target loci comprise one or more single nucleotide polymorphisms (SNPs) and wherein the estimating comprises using allele calls at the SNPs.
8. The method of claim 7 , wherein the measuring comprises quantitative allele measurements.
9. The method of claim 8 , wherein the quantitative allele measurements are used for determining the ploidy state and for estimating the fraction of fetal cell-free DNA in the maternal blood sample.
10. The method of claim 2 , wherein one or more of the target loci map to a reference chromosome.
11. The method of claim 10 , wherein the target loci comprise one or more single nucleotide polymorphisms (SNPs).
12. The method of claim 11 , wherein a Z-score is determined using measurements of the amounts of target loci on the target chromosome and the reference chromosome, and the Z-score is used to determine the ploidy state of the target chromosome.
13. The method of claim 1 , wherein the multiplex PCR comprises amplifying at least 2,000 SNP target loci from the cell-free DNA in one reaction mixture.
14. The method of claim 1 , wherein the multiplex PCR comprises amplifying at least 5,000 SNP target loci from the cell-free DNA in one reaction mixture.