Droplet Microfluidics for the Label-Free Extraction of Complete Phase Diagrams and Kinetics of Liquid-Liquid Phase Separation in Finite Volumes.

Liquid-liquid phase separation of polymer and protein solutions is central in many areas of biology and material sciences. Here, an experimental and theoretical framework is provided to investigate the thermodynamics and kinetics of liquid-liquid phase separation in volumes comparable to cells. The strategy leverages droplet microfluidics to accurately measure the volume of the dense phase generated by liquid-liquid phase separation of solutions confined in micro-sized compartments. It is shown that the measurement of the volume fraction of the dense phase at different temperatures allows the evaluation of the binodal lines that determine the coexistence region of the two phases in the temperature-concentration phase diagram. By applying a thermodynamic model of phase separation in finite volumes, it is further shown that the platform can predict and validate kinetic barriers associated with the formation of a dense droplet in a parent dilute phase, therefore connecting thermodynamics and kinetics of liquid-liquid phase separation.

Added Value of Clinical Sequencing: WGS-Based Profiling of Pharmacogenes.

Although several pharmacogenetic (PGx) predispositions affecting drug efficacy and safety are well established, drug selection and dosing as well as clinical trials are often performed in a non-pharmacogenetically-stratified manner, ultimately burdening healthcare systems. Pre-emptive PGx testing offers a solution which is often performed using microarrays or targeted gene panels, testing for common/known PGx variants. However, as an added value, whole-genome sequencing (WGS) could detect not only disease-causing but also pharmacogenetically-relevant variants in a single assay. Here, we present our WGS-based pipeline that extends the genetic testing of Mendelian diseases with PGx profiling, enabling the detection of rare/novel PGx variants as well. From our in-house WGS (PCR-free 60× PE150) data of 547 individuals we extracted PGx variants with drug-dosing recommendations of the Dutch Pharmacogenetics Working Group (DPWG). Furthermore, we explored the landscape of DPWG pharmacogenes in gnomAD and our in-house cohort as well as compared bioinformatic tools for WGS-based structural variant detection in CYP2D6. We show that although common/known PGx variants comprise the vast majority of detected DPWG pharmacogene alleles, for better precision medicine, PGx testing should move towards WGS-based approaches. Indeed, WGS-based PGx profiling is not only feasible and future-oriented but also the most comprehensive all-in-one approach without generating significant additional costs.

Parallel multi-droplet platform for reaction kinetics and optimization.

We present an automated droplet reactor platform possessing parallel reactor channels and a scheduling algorithm that orchestrates all of the parallel hardware operations and ensures droplet integrity as well as overall efficiency. We design and incorporate all of the necessary hardware and software to enable the platform to be used to study both thermal and photochemical reactions. We incorporate a Bayesian optimization algorithm into the control software to enable reaction optimization over both categorical and continuous variables. We demonstrate the capabilities of both the preliminary single-channel and parallelized versions of the platform using a series of model thermal and photochemical reactions. We conduct a series of reaction optimization campaigns and demonstrate rapid acquisition of the data necessary to determine reaction kinetics. The platform is flexible in terms of use case: it can be used either to investigate reaction kinetics or to perform reaction optimization over a wide range of chemical domains.

Potential of whole-genome sequencing-based pharmacogenetic profiling.

Pharmacogenetics represents a major driver of precision medicine, promising individualized drug selection and dosing. Traditionally, pharmacogenetic profiling has been performed using targeted genotyping that focuses on common/known variants. Recently, whole-genome sequencing (WGS) is emerging as a more comprehensive short-read next-generation sequencing approach, enabling both gene diagnostics and pharmacogenetic profiling, including rare/novel variants, in a single assay. Using the example of the pharmacogene CYP2D6, we demonstrate the potential of WGS-based pharmacogenetic profiling as well as emphasize the limitations of short-read next-generation sequencing. In the near future, we envision a shift toward long-read sequencing as the predominant method for gene diagnostics and pharmacogenetic profiling, providing unprecedented data quality and improving patient care.