Non-invasive fetal genotyping for maternal alleles with droplet digital PCR: A comparative study of analytical approaches

Objectives

To develop a flexible droplet digital PCR (ddPCR) workflow to perform non-invasive prenatal diagnosis via relative mutation dosage (RMD) for maternal pathogenic variants with a range of inheritance patterns, and to compare the accuracy of multiple analytical approaches.

Methods

Cell free DNA (cfDNA) was tested from 124 archived maternal plasma samples: 88 cases for sickle cell disease and 36 for rare Mendelian conditions. Three analytical methods were compared: sequential probability ratio testing (SPRT), Bayesian and z-score analyses.

Results

The SPRT, Bayesian and z-score analyses performed similarly well with correct prediction rates of 96%, 97% and 98%, respectively. However, there were high rates of inconclusive results for each cohort, particularly for z-score analysis which was 31% overall. Two samples were incorrectly classified by all three analytical methods; a false negative result predicted for a fetus affected with sickle cell disease and a false positive result predicting the presence of an X-linked IDS variant in an unaffected fetus.

Conclusions

ddPCR can be applied to RMD for diverse conditions and inheritance patterns, but all methods carry a small risk of erroneous results. Further evaluation is required both to reduce the rate of inconclusive results and explore discordant results in more detail.     

The Role of Headwater Streams in Downstream Water Quality1

Abstract: Knowledge of headwater influences on the water‐quality and flow conditions of downstream waters is essential to water‐resource management at all governmental levels; this includes recent court decisions on the jurisdiction of the Federal Clean Water Act (CWA) over upland areas that contribute to larger downstream water bodies. We review current watershed research and use a water‐quality model to investigate headwater influences on downstream receiving waters. Our evaluations demonstrate the intrinsic connections of headwaters to landscape processes and downstream waters through their influence on the supply, transport, and fate of water and solutes in watersheds. Hydrological processes in headwater catchments control the recharge of subsurface water stores, flow paths, and residence times of water throughout landscapes. The dynamic coupling of hydrological and biogeochemical processes in upland streams further controls the chemical form, timing, and longitudinal distances of solute transport to downstream waters. We apply the spatially explicit, mass‐balance watershed model SPARROW to consider transport and transformations of water and nutrients throughout stream networks in the northeastern United States. We simulate fluxes of nitrogen, a primary nutrient that is a water‐quality concern for acidification of streams and lakes and eutrophication of coastal waters, and refine the model structure to include literature observations of nitrogen removal in streams and lakes. We quantify nitrogen transport from headwaters to downstream navigable waters, where headwaters are defined within the model as first‐order, perennial streams that include flow and nitrogen contributions from smaller, intermittent and ephemeral streams. We find that first‐order headwaters contribute approximately 70% of the mean‐annual water volume and 65% of the nitrogen flux in second‐order streams. Their contributions to mean water volume and nitrogen flux decline only marginally to about 55% and 40% in fourth‐ and higher‐order rivers that include navigable waters and their tributaries. These results underscore the profound influence that headwater areas have on shaping downstream water quantity and water quality. The results have relevance to water‐resource management and regulatory decisions and potentially broaden understanding of the spatial extent of Federal CWA jurisdiction in U.S. waters.