Haplotyping germline and cancer genomes with high-throughput linked-read sequencing.

Haplotyping of human chromosomes is a prerequisite for cataloguing the full repertoire of genetic variation. We present a microfluidics-based, linked-read sequencing technology that can phase and haplotype germline and cancer genomes using nanograms of input DNA. This high-throughput platform prepares barcoded libraries for short-read sequencing and computationally reconstructs long-range haplotype and structural variant information. We generate haplotype blocks in a nuclear trio that are concordant with expected inheritance patterns and phase a set of structural variants. We also resolve the structure of the EML4-ALK gene fusion in the NCI-H2228 cancer cell line using phased exome sequencing. Finally, we assign genetic aberrations to specific megabase-scale haplotypes generated from whole-genome sequencing of a primary colorectal adenocarcinoma. This approach resolves haplotype information using up to 100 times less genomic DNA than some methods and enables the accurate detection of structural variants.

Vector separation of particles and cells using an array of slanted open cavities.

We present a microfluidic platform for the continuous separation of suspended particles based on their size and settling velocity. The separation method takes advantage of the flow field in the vicinity and inside slanted open cavities. These cavities induce flow along them, which deflects the suspended particles to a different degree depending on the extent to which they penetrate into the cavities. The cumulative deflection in the periodic array ultimately leads to vector chromatography, with the different species in the sample moving in different directions. We demonstrate density and size based separation over a range of flow rates by separating polystyrene and silica particles and show that purities nearing 100% can be achieved for multicomponent mixtures. We also demonstrate the potential of the platform to separate biological cells by fractionating different blood components. We discuss the presence of two regimes, depending on the ratio between the settling velocity and the velocity of the particles across the open cavities. The proposed platform could also integrate additional separative force fields in the direction normal to the plane of the cavities to fractionate specific mixtures based on the distinguishing properties of the component species.

Stochastic and deterministic vector chromatography of suspended particles in one-dimensional periodic potentials.

We present a comprehensive description of vector chromatography (VC) that includes deterministic and stochastic transport in one-dimensional periodic free-energy landscapes, with both energetic and entropic contributions, and identifies the parameters governing the deflection angle. We also investigate the dependence of the deflection angle on the shape of the free-energy landscape by varying the width of the linear transitions in an otherwise dichotomous potential. Finally, we present experimental results obtained in a microfluidic system in which gravity drives the suspended particles and, in combination with a bottom surface patterned with shallow rectangular grooves, creates a periodic landscape of (potential) energy barriers. The experiments validate the model and demonstrate that a simple, passive microdevice can lead to VC of colloidal particles based on both size and density. More generally, other fields, e.g., electric, dielectrophoretic, or magnetic, can play or enhance the role of gravity, potentially leading to a versatile technique.

Partition-induced vector chromatography in microfluidic devices.

We investigate by means of macrotransport theory the transport of Brownian particles in a slit geometry in the presence of an arbitrary two-dimensional periodic energy landscape and driven by an external force or convected by a flow field. We obtained analytical expressions for the probability distribution and the average migration angle of the particles under the Fick-Jacobs approximation. The migration angle is shown to differ from the angle of the driving field and to strongly depend on the physical properties of the suspended species, thus providing the basis for vector chromatography, in which different species move in different directions and can be continuously fractionated. The potential of microfluidic devices as a platform for partition-induced vector chromatography is demonstrated by considering the particular case of a piece-wise constant, periodic potential that, in equilibrium, induces the spontaneous partition of different species into high and low concentration stripes, and which can be easily fabricated by patterning physically or chemically one of the surfaces of a channel. We show the feasibility to fractionate a mixture of particles for systems in which partition is induced via 1-g gravity and Van der Waals interactions in physically or chemically patterned channels.