Figure 2.

(A) (Top) Distinguishing an affected (homozygous) fetus from mock unaffected fetus (negative controls) by a calculated empirical Z-score (Equation S2) based on allelic count differences of each separate position. Measurement of the alleles on the mutation site directly (leftmost column) and by a multiplex amplification of 10 additional positions (right) that are linked to the mutation through a known 1.7 Mbp haplotype. (Bottom) Location of mutations and haplotype positions relative to the MUT gene and Chromosome 6.

(B) Determination of fetal fraction by tallying allelic counts of a panel of blindly queried SNVs that are diversely represented in the human population. By finding SNP positions where the mother is homozygous and the fetus is heterozygous (ie AA/AG), fetal fraction can be calculated to be double the fractional count of the alternative allele in the fetus (ie 2 times the fraction count of G). To be almost guaranteed multiple useful positions that meet the criteria for fetal fraction determination, we screened the maternal cell portion against 32 SNP positions. Positions that were homozygous and had high probe quality tallied 13. These corresponding 13 probes were pooled for a multiplex PCR of the plasma DNA (7200 input molecules per position). Calculation of a minor allele fraction, which is the smaller fraction of the two counted alleles and half of the fetal fraction, helped to determine which of the 13 positions were useful. Three positions: rs13218440, rs12423234, rs1821380 had a fetal fraction of 16.7%, 15.4%, and 17.8% respectively.