Simultaneous determination of aneuploidy and fetal fraction

A method for simultaneously determining fetal aneuploidy and fetal fraction in a maternal plasma, serum, or urine sample comprising an unamplified mixture of fetal and maternal cell-free DNA (cfDNA) nucleic acid molecules, said method comprising:

• (a) enriching a portion of said unamplified mixture for a plurality of polymorphic target nucleic acids, wherein said enriching comprises:
• (i) dividing said unamplified mixture into a first portion and a second portion, and specifically amplifying said plurality of target nucleic acids in the first portion using primer pairs each capable of amplifying a target nucleic acid sequence comprising a polymorphic site in a multiplex PCR reaction to generate an amplified product comprising a panel of amplified polymorphic sites that contains a sufficient number of polymorphic sites such that at least two are informative polymorphic sites;
• and
• (ii) combining at least a portion or all of the amplified product with at least a portion of the second portion of said unamplified mixture to obtain an enriched mixture;
• (b) performing massively parallel sequencing of at least a portion of the enriched mixture obtained in step (a), wherein said sequencing comprises providing a plurality of sequence reads and aligning each sequence read to a chromosome in a reference genome to identify a plurality of sequence tags;
• and
• (c) based on said sequencing, simultaneously determining said fetal fraction and said aneuploidy, wherein:
• (i) determining said fetal fraction is performed by a processor using computer readable software code and comprises:
• (1) using the mapped sequence tags to identify at least two informative polymorphic sites in said panel of amplified polymorphic sites;
• and
• (2) calculating the fetal fraction based on the total number of mapped sequence tags that map to a first allele and the total number of mapped sequence tags that map to a second allele at each of said informative polymorphic sites;
• and
• (ii) determining said fetal aneuploidy is performed by a processor using computer readable software code and comprises quantification of the number of mapped sequence tags aligning to a chromosome of interest, and comparing the results obtained for the chromosome of interest to a threshold value, wherein the threshold value is a number that serves as a limit of diagnosis of an aneuploidy and wherein the presence of an aneuploidy for the chromosome of interest is identified if the threshold value is exceeded by the results obtained for the chromosome of interest.