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 amplifying target loci in a nucleic acid sample, the method comprising:
contacting the nucleic acid sample comprising target loci with a library of at least 10 primers that simultaneously hybridize to at least 10 target loci to produce a reaction mixture;
subjecting the reaction mixture to primer extension reaction conditions to produce amplified products comprising target amplicons, wherein concentration of each primer is 20 nM or less, wherein length of an annealing step of the reaction conditions is 10 minutes or more, and wherein the at least target loci are simultaneously amplified; and
sequencing the amplified products comprising the target amplicons.
2 . The method of claim 1 , wherein the library comprises at least 50 primer pairs that simultaneously hybridize to at least 50 target loci, and wherein the at least 50 target loci are simultaneously amplified.
3 . The method of claim 1 , wherein the length of the annealing step is between 10 and 20 minutes.
4 . The method of claim 1 , wherein the concentration of each primer in the library is 10 nM or less.
5 . The method of claim 1 , wherein the annealing step comprises annealing at a temperature of 60° C. to 65° C.
6 . The method of claim 1 , wherein at least 80% of the amplified products are target amplicons.
7 . The method of claim 1 , wherein at least 90% of the amplified products are target amplicons.
8 . The method of claim 1 , wherein length of each target amplicon is less than 100 bp.
9 . The method of claim 1 , wherein length of each target amplicon is 60-80 bp.
10 . The method of claim 1 , wherein the target loci each comprises a single nucleotide polymorphism (SNP).
11 . The method of claim 1 , wherein the nucleic acid sample comprises nucleic acids from a mixed origin.
12 . The method of claim 11 , wherein the nucleic acid sample comprises nucleic acids from a tumor.