Second generation noninvasive fetal genome analysis reveals de novo mutations, single-base parental inheritance, and preferred DNA ends

Abstract

Plasma DNA obtained from a pregnant woman was sequenced to a depth of 270× haploid genome coverage. Comparing the maternal plasma DNA sequencing data with the parental genomic DNA data and using a series of bioinformatics filters, fetal de novo mutations were detected at a sensitivity of 85% and a positive predictive value of 74%. These results represent a 169-fold improvement in the positive predictive value over previous attempts. Improvements in the interpretation of the sequence information of every base position in the genome allowed us to interrogate the maternal inheritance of the fetus for 618,271 of 656,676 (94.2%) heterozygous SNPs within the maternal genome. The fetal genotype at each of these sites was deduced individually, unlike previously, where the inheritance was determined for a collection of sites within a haplotype. These results represent a 90-fold enhancement in the resolution in determining the fetus's maternal inheritance. Selected genomic locations were more likely to be found at the ends of plasma DNA molecules. We found that a subset of such preferred ends exhibited selectivity for fetal- or maternal-derived DNA in maternal plasma. The ratio of the number of maternal plasma DNA molecules with fetal preferred ends to those with maternal preferred ends showed a correlation with the fetal DNA fraction. Finally, this second generation approach for noninvasive fetal whole-genome analysis was validated in a pregnancy diagnosed with cardiofaciocutaneous syndrome with maternal plasma DNA sequenced to 195× coverage. The causative de novo BRAF mutation was successfully detected through the maternal plasma DNA analysis.

Keywords: DNA fragmentation patterns; massively parallel sequencing; noninvasive prenatal testing.

Fig. 1.

Summary of the key differences between the first and second generation approaches for noninvasive fetal whole-genome analysis. ^aThe performance characteristics quoted for first generation fetal whole-genome analysis were from refs. –. *RHDO represents RHDO analysis, and GRAD represents GRAD analysis.

Fig. S1.

Size distribution of plasma DNA fragments carrying fetal-specific alleles (blue) and alleles shared by the fetus and the mother (red).

Fig. S2.

Frequency plot of the size difference between plasma DNA fragments carrying fetal-specific alleles and alleles shared by the fetus and the mother at SNPs where the mother was homozygous and the fetus was heterozygous for the third trimester pregnancy case. The red dotted line indicates the size difference observed in 90% of the SNPs. (A) Third trimester case. (B) The case carrying a fetus with cardiofaciocutaneous syndrome.

Fig. 2.

Detection of the fetal de novo mutations through the analysis of the sequencing data of maternal plasma DNA. The number in bold in each step represents the number of candidate de novo mutations identified after the particular process.

Fig. S3.

Simulation analysis for the number of SNPs per one accurate GRAD classification of maternal inheritance for different fetal DNA fractions and sequencing depths.

Fig. 3.

Left shows an illustrative example of the nonrandom fragmentation patterns of plasma DNA carrying a fetal-specific allele and an allele shared by the mother and the fetus. In Upper, each horizontal line represents one sequenced DNA fragment. The ends of the DNA fragments represent the ending position of the sequenced read. The fragments are sorted according to the coordinate of the left outermost nucleotide (genomic coordinate with the lower numerical value). In Lower, the percentage of fragments ending on a particular position is shown. Upper Right shows the span of all of the sequenced DNA fragments aligned to the same SNP obtained from the sonicated blood cell DNA of the same pregnant woman. Lower Right shows the percentage of fragments ending on a particular position. The x axes show the relative genomic coordinates. The SNP of interest is located in the middle (marked by a dotted line).

Fig. 4.

Plot of probability of a genomic coordinate being an ending position of maternal plasma DNA fragments across a region with an informative SNP at which (A) the mother was homozygous and the fetus was heterozygous and (B) the mother was heterozygous and the fetus was homozygous. (A) Results for nucleotide positions with a significantly increased probability of being an end of plasma DNA fragments carrying a shared allele and a fetal-specific allele are shown in red and blue, respectively. (B) Results for nucleotide positions with a significantly increased probability of being an end of plasma DNA fragments carrying a shared allele and a maternal-specific allele are shown in red and blue, respectively. The x axes of the graphs show the relative genomic coordinates. The position of the SNP of interest is marked by a dotted line.

Fig. 5.

Analysis of ending positions for plasma DNA fragments across (A) SNPs that were homozygous in the mother and heterozygous in the fetus and (B) SNPs that were homozygous in the fetus and heterozygous in the mother. (A) Set A included preferred ending positions for fragments carrying fetal-specific alleles. Set B included preferred ending positions for fragments carrying shared alleles. Set C included preferred ending positions for both types of plasma DNA fragments. (B) Set X included preferred ending positions for fragments carrying maternal-specific alleles. Set Y included preferred ending positions for fragments carrying shared alleles. Set Z included preferred ending positions for both types of plasma DNA fragments.

Fig. 6.

Correlation between the relative abundance (ratio F/M) of plasma DNA molecules with preferred fetal (Set A) and maternal (Set X) ends, and fetal DNA fraction.

Fig. 7.

(A) Plasma DNA size distributions for fragments ending on fetal-preferred ending positions (Set A; blue) and fragments ending on maternal-preferred ending positions (Set X; red). A shorter size distribution was observed for fragments ending on Set A positions compared with those ending on Set X positions. (B) Cumulative plot for the size distributions for the two sets of fragments. The difference in the cumulative frequencies of the two sets of fragments (ΔS) against fragment size is shown. (C) A plot of ΔS against size when the end coordinates are shifted by 0 to +5 bp with regard to the genomic coordinates of the preferred end. (D) A plot of ΔS against size when the end coordinates are shifted by 0 to −5 bp with regard to the genomic coordinates of the preferred end.

Fig. 8.

(A) Plasma DNA size distributions in a pooled plasma DNA sample from 26 first trimester pregnant women for fragments ending on fetal-preferred ending positions (Set A; blue) and fragments ending on maternal-preferred ending positions (Set X; red). A shorter size distribution was observed for fragments ending on Set A positions compared with those ending on Set X positions. (B) Cumulative plot for the size distributions for the two sets of fragments. The difference in the cumulative frequencies of the two sets of fragments (ΔS) against fragment size is shown. (C) A plot of ΔS against size when the end coordinates are shifted by 0 to +5 bp with regard to the genomic coordinates of the preferred end. (D) A plot of ΔS against size when the end coordinates are shifted by 0 to −5 bp with regard to the genomic coordinates of the preferred end.

Fig. S4.

Detection of the fetal de novo mutations through the analysis of the sequencing data of maternal plasma DNA for the case with cardiofaciocutaneous syndrome. The number in bold in each step represents the number of candidate de novo mutations identified after the particular process.

Fig. S5.

Analysis of ending positions for plasma DNA fragments across (A) SNPs that were homozygous in the mother and heterozygous in the fetus and (B) SNPs that were homozygous in the fetus and heterozygous in the mother for the case with cardiofaciocutaneous syndrome. (A) Set A included preferred ending positions for fragments carrying fetal-specific alleles. Set B included preferred ending positions for fragments carrying shared alleles. Set C included preferred ending positions for both types of plasma DNA fragments. (B) Set X included preferred ending positions for fragments carrying maternal-specific alleles. Set Y included preferred ending positions for fragments carrying shared alleles. Set Z included preferred ending positions for both types of plasma DNA fragments.

Fig. S6.

Correlation between the relative abundance (ratio F/M) of plasma DNA molecules with preferred fetal (Set A) and maternal (Set X) ends obtained from the case with cardiofaciocutaneous syndrome and fetal DNA fraction.