fig1

Figure 1. NIPD technologies for the detection of paternally inherited or de novo variants. Technologies developed for the detection of low-level paternally inherited or de novo variants. A: Real-time-quantitative polymerase chain reaction (RT-qPCR) determines the presence of a particular genomic target sequence above a PCR cycle threshold (Ct) set using a negative control. B: PCR followed by restriction enzyme digestion (PCR-RED) is applicable where the variant of interest creates a novel restriction enzyme (RE) recognition site otherwise absent in the wildtype (WT) target sequence. A typical negative control is maternal gDNA, while cfDNA and gDNA positive controls can also be run alongside. The digestion products are visualised by gel electrophoresis, where the presence of a lower molecular weight (Mr) band is indicative of a positive result (indicated by the red box in the diagram). C: Co-amplification at lower denaturation temperature-PCR (COLD-PCR) is applicable for targets where the variant allele creates a lower denaturation temperature than the WT allele, and can therefore be preferentially amplified using a modified PCR protocol (i.e., with a lower denaturation temperature during cycling). The variant-enriched product is detectable via Sanger sequencing or next-generation sequencing (NGS). D: Digital PCR (dPCR) is carried out using microfluidic devices or oil-water-emulsion technology (ddPCR) for compartmentalisation of individual genomic target sequences within nanolitre-sized chambers or droplets, respectively. Fluorescently labelled probes are designed with sequence homology to the target of interest such that recognition and binding generates a signal indicative of the presence of the target within the cfDNA sample. This signal can be quantified by counting the number of positive reaction units relative to the total number of reaction units containing a DNA molecule. E: PCR followed by next-generation sequencing (PCR-NGS) involves the use of targeted PCR for the enrichment of a short target region of interest (< 10 kb), followed by short-read sequencing at > 10,000X read depth (~150 bp per read). F: Capture-based NGS involves the use of targeted probes for the enrichment of a desired genomic locus (up to several Mb), followed by short-read sequencing to ~100X read depth for accurate variant detection without PCR-related artefacts.