Noninvasive prenatal diagnosis of fetal trisomy by allelic ratio analysis using targeted massively parallel sequencing

A method of analyzing a biological sample from a female subject pregnant with a fetus to determine whether the fetus has an aneuploidy associated with a first chromosome, the biological sample containing a mixture of cell-free DNA molecules from the fetus and the female subject, the method comprising:

• enriching the biological sample for DNA molecules from a plurality of target regions that are known to have polymorphic loci;
• after the enriching, sequencing the DNA molecules from the biological sample to obtain a plurality of sequence reads;
• analyzing, by a computer system, the plurality of sequence reads, wherein analyzing a sequence read includes:
• identifying a location of the sequence read in a reference genome by aligning the sequence read to the reference genome;
• and
• determining a respective allele of the sequence read;
• identifying, by the computer system, a plurality of first loci on the first chromosome, the plurality of first loci corresponding to a portion of the target regions;
• identifying, by the computer system, a plurality of second loci on one or more reference chromosomes, the plurality of second loci corresponding to a portion of the target regions, wherein the pregnant female is homozygous for a respective maternal allele at each of the first and second loci, and wherein the fetus is heterozygous for the respective maternal allele and a respective paternal allele at each of the first and second loci, the respective paternal alleles being different from the respective maternal alleles;
• determining, by the computer system, a first maternal amount of the respective maternal alleles at the plurality of first loci;
• determining, by the computer system, a first paternal amount of the respective paternal alleles at the plurality of first loci;
• calculating a first ratio of the first maternal amount and the first paternal amount;
• determining, by the computer system, a second maternal amount of the respective maternal alleles at the plurality of second loci;
• determining, by the computer system, a second paternal amount of the respective paternal alleles at the plurality of second loci;
• calculating a second ratio of the second maternal amount and the second paternal amount;
• calculating a third ratio of the first ratio and the second ratio;
• and
• comparing the third ratio to one or more cutoff values to determine whether the fetus has an aneuploidy associated with the first chromosome, wherein a first cutoff value of the one or more cutoff values corresponds to an aneuploidy that is paternally-derived.