Genome-Wide Sequencing for Prenatal Detection of Fetal Single-Gene Disorders

Abstract

New sequencing methods capable of rapidly analyzing the genome at increasing resolution have transformed diagnosis of single-gene or oligogenic genetic disorders in pediatric and adult medicine. Targeted tests, consisting of disease-focused multigene panels and diagnostic exome sequencing to interrogate the sequence of the coding regions of nearly all genes, are now clinically offered when there is suspicion for an undiagnosed genetic disorder or cancer in children and adults. Implementation of diagnostic exome and genome sequencing tests on invasively and noninvasively obtained fetal DNA samples for prenatal genetic diagnosis is also being explored. We predict that they will become more widely integrated into prenatal care in the near future. Providers must prepare for the practical, ethical, and societal dilemmas that accompany the capacity to generate and analyze large amounts of genetic information about the fetus during pregnancy.

Figure 1.

Workflow for next-generation sequencing (NGS). (A) Library preparation: Genomic DNA is prepared from a prenatal sample (amniotic fluid or chorionic villi) and fragmented. This is followed by adapter ligation and preparation of the sequencing library. When WGS is performed, the library is directly denatured for use as sequencing templates (blue lines and arrows). For targeted approaches such as whole-exome sequencing (purple lines and arrows), an additional step to capture and enrich for fragments of interest (such as coding exons) for sequencing by hybridizing them to a library of baits with known sequence, followed by purification and bait release is required. (B) Sequencing procedure: Fragments are immobilized by hybridization to linkers (here, represented on a solid surface, but other methods exist), followed by multiple rounds of replication and clonal amplification. Differently fluorescently labeled nucleotides are then added to the single-stranded templates and emitting light is used to identify the added nucleotide that is complementary to the template. The sequenced fragments are then aligned to the reference genome sequence in multiple copies (sequencing reads). In WGS, the alignment covers all regions for which sequencing was successfully sequenced. In targeted sequencing, such as WES, only those sequences represented in the baits are covered, typically with more reads for each.