Non-invasive prenatal measurement of the fetal genome

Abstract

The vast majority of prenatal genetic testing requires invasive sampling. However, this poses a risk to the fetus, so one must make a decision that weighs the desire for genetic information against the risk of an adverse outcome due to hazards of the testing process. These issues are not required to be coupled, and it would be desirable to discover genetic information about the fetus without incurring a health risk. Here we demonstrate that it is possible to non-invasively sequence the entire prenatal genome. Our results show that molecular counting of parental haplotypes in maternal plasma by shotgun sequencing of maternal plasma DNA allows the inherited fetal genome to be deciphered non-invasively. We also applied the counting principle directly to each allele in the fetal exome by performing exome capture on maternal plasma DNA before shotgun sequencing. This approach enables non-invasive exome screening of clinically relevant and deleterious alleles that were paternally inherited or had arisen as de novo germline mutations, and complements the haplotype counting approach to provide a comprehensive view of the fetal genome. Non-invasive determination of the fetal genome may ultimately facilitate the diagnosis of all inherited and de novo genetic disease.

Figure 1

Molecular counting strategies for measuring the fetal genome noninvasively from maternal blood only. Genome-wide, chromosome length haplotypes of the mother are obtained using direct deterministic phasing. The inheritance of maternal haplotypes is revealed by sequencing maternal plasma DNA and summing the count of the alleles specific to each haplotype at heterozygous loci and determining the relative representation of the two alleles. The inherited paternal haplotypes are defined by the paternal specific alleles (i.e. those that are different from the maternal ones at positions where the mother is homozygous). The allelic identity at loci linked to the paternal specific alleles on the paternal haplotype can be imputed. Alternatively, molecular counting can be applied directly to count alleles at individual locus to determine fetal genotypes via targeted deep sequencing, such as exome enriched sequencing of maternal plasma DNA. For illustrative purpose, each locus is biallelic and carries the ‘A’ or ‘G’ alleles.

Figure 2

Noninvasively determining genome-wide fetal inheritance of maternal haplotypes via haplotype counting of maternal plasma DNA with at least 99.8% accuracy over 99% of the genome in three maternal plasma samples (A-C). Each point on a black line represents the relative amount of the two maternal haplotypes evaluated using the markers lying within a bin centered at the point, and is accompanied by a white bar that corresponds to the 95% confidence interval for each measurement. The maternal haplotypes are colored pink or grey according to the true transmission states, as determined by fetal cord blood genotypes. Over-representation of ‘maternal haplotype 2’ in P2T3 maternal plasma immediately adjacent to the DiGeorge syndrome associated deletion (blue) indicates fetal inheritance of the deletion, which agrees with fetal cord blood genotype.

Figure 3

Reconstruction of paternally inherited chromosomes noninvasively based on imputation using observed non-maternal alleles. The paternally inherited haplotypes were reconstructed by detection of paternal specific alleles, followed by imputation at linked positions. At the final sequencing depth, ~66–70% of all the paternal specific alleles were detected at least once. Using those markers, ~70% of the paternally inherited haplotypes were imputed with ~94–97% accuracy. The loci that could not be confidently imputed could in principle be completely determined by deeper sequencing and application of the counting principle directly to the individual alleles at every genomic position.

Figure 4

Exome sequencing of P1 maternal plasma DNA in all three trimesters to determine maternal and fetal genotypes.. A-C. Histograms of minor allele fraction in maternal plasma from all three trimesters of P1 at positions that are confidently called in both plasma sequencing data and pure fetal/maternal DNA genotyping data. Insets: ROC curves of positions detecting fetal genotypes differing from maternal genotype when the maternal position is either homozygous or heterozygous. The higher the fetal fraction (~6, 20, 26% for Trimester 1–3), the more the distributions are separated, and the easier it is to distinguish between the two distributions of fetal genotype. D. Histogram of per-position coverage, with bin size of 5. Exome positions >100X are 75%, 78%, and 90% respectively for Trimester 1–3 and >200X are 48%, 56%, and 84%. E-F. ROCs curves at genomic positions where mother is heterozygous (E) or homozygous (F), using either sequencing or SNP array of pure DNA as references for maternal and fetal genotypes. ‘SeqRef’ uses a sequenced reference, ‘Array’ uses a SNP array, and ‘SeqRef-Array’ uses a sequenced reference only at positions on a SNP array.