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 amplifying target loci from a maternal blood sample from a pregnant mother of a fetus, the method comprising:
a) extracting DNA from the maternal blood sample, wherein the DNA comprises cell-free DNA from the pregnant mother and from the fetus, comprising 50 to 5000 target loci comprising polymorphic loci and non-polymorphic loci; and
b) enriching the extracted DNA at the target loci by:
i) hybridizing an upstream and a downstream ligation-mediated PCR probe to one strand of the cell free DNA within a region of DNA that comprises a target locus for each of the target loci,
ii) ligating an upstream and a downstream ligation-mediated PCR probe that are hybridized to the same region of DNA comprising a target locus; and
iii) amplifying ligated ligation-mediated PCR probes using PCR, thereby amplifying the target loci.
2 . The method of claim 1 , wherein the method further comprises detecting the amplified target loci using a microarray.
3 . The method of claim 1 , wherein the target loci comprise between 50 and 1000 loci.
4 . The method of claim 1 , wherein the polymorphic loci and the non-polymorphic loci are amplified in a single reaction.
5 . The method of claim 1 , wherein the target loci comprise loci from a target chromosome and loci from a reference chromosome presumed to be diploid in both the mother and the fetus.
6 . The method of claim 5 , wherein the target chromosome is chromosome X, Y, 13, 18, and/or 21, and the reference chromosome comprises chromosome 1 and/or 2.
7 . The method of claim 1 , wherein the method further comprises measuring an amount of fetal DNA and an amount of maternal DNA in the maternal blood sample.
8 . The method of claim 1 , wherein the method further comprises measuring an amount of one or more alleles at the target loci.
9 . The method of claim 1 , wherein each of the upstream and the downstream ligation-mediated PCR probe hybridize to one strand of the cell free DNA within a region of DNA that is separated by 60 nucleotides or less from a target locus for each of the target loci.
10 . The method of claim 1 , wherein the DNA is free-floating DNA isolated from plasma.
11 . A method for measuring the amount of one or more alleles at target loci in a maternal blood sample from a pregnant mother of a fetus, comprising:
a) extracting DNA from the maternal blood sample, wherein the DNA comprises cell-free DNA from the pregnant mother and from the fetus, wherein the target loci comprise 50 to 5000 polymorphic loci and non-polymorphic loci;
b) preparing a DNA fragment library with universal adaptors on both ends of DNA fragments of the DNA fragment library using the extracted DNA, wherein the DNA fragments comprise the target loci:
c) amplifying the DNA fragments using universal primers that bind to the universal adapters on the ends of the DNA fragments;
d) detecting the amplified DNA fragments using a microarray; and
e) measuring an amount of one or more alleles at the target loci using the amount of detected amplified DNA fragments.
12 . The method of claim 11 , wherein the preparing the DNA fragment library with universal adaptors comprises:
a) hybridizing an upstream and a downstream ligation-mediated PCR probe to one strand of the cell free DNA within a region of DNA comprises a target locus for each of the target loci, and
b) ligating an upstream and a downstream ligation-mediated PCR probe that are hybridized to the same region of DNA comprising a target locus.
13 . The method of claim 12 , wherein each of the upstream and the downstream ligation-mediated PCR probes hybridize to one strand of the cell free DNA within a region of DNA that is separated by 60 nucleotides or less from a target locus for each of the target loci.
14 . The method of claim 11 , wherein the target loci comprise between 50 and 1000 loci.
15 . The method of claim 12 , wherein the polymorphic loci and the non-polymorphic loci are amplified in a single reaction.
16 . The method of claim 11 , wherein the target loci comprise loci from a target chromosome and loci from a reference chromosome presumed to be diploid in both the mother and the fetus.
17 . The method of claim 16 , wherein the target chromosome is chromosomes X, Y, 13, 18, and/or 21, and the reference chromosome comprises chromosome 1 and/or 2.
18 . The method of claim 11 , wherein the method further comprises measuring an amount of fetal DNA and an amount of maternal DNA in the maternal blood sample.
19 . The method of claim 11 , wherein at least some of the target loci comprise single nucleotide polymorphisms.
20 . The method of claim 11 , wherein the DNA is free-floating DNA isolated from plasma.