Kevin Ness

High-Throughput Droplet Digital PCR System for Absolute Quantitation of DNA Copy Number

Digital PCR enables the absolute quantitation of nucleic acids in a sample. The lack of scalable and practical technologies for digital PCR implementation has hampered the widespread adoption of this inherently powerful technique. Here we describe a high-throughput droplet digital PCR (ddPCR) system that enables processing of ∼2 million PCR reactions using conventional TaqMan assays with a 96-well plate workflow. Three applications demonstrate that the massive partitioning afforded by our ddPCR system provides orders of magnitude more precision and sensitivity than real-time PCR. First, we show the accurate measurement of germline copy number variation. Second, for rare alleles, we show sensitive detection of mutant DNA in a 100 000-fold excess of wildtype background. Third, we demonstrate absolute quantitation of circulating fetal and maternal DNA from cell-free plasma. We anticipate this ddPCR system will allow researchers to explore complex genetic landscapes, discover and validate new disease associations, and define a new era of molecular diagnostics.

Massively parallel digital transcriptional profiling of single cells

Characterizing the transcriptome of individual cells is fundamental to understanding complex biological systems. We describe a droplet-based system that enables 3′ mRNA counting of tens of thousands of single cells per sample. Cell encapsulation, of up to 8 samples at a time, takes place in ∼6 min, with ∼50% cell capture efficiency. To demonstrate the systems technical performance, we collected transcriptome data from ∼250k single cells across 29 samples. We validated the sensitivity of the system and its ability to detect rare populations using cell lines and synthetic RNAs. We profiled 68k peripheral blood mononuclear cells to demonstrate the systems ability to characterize large immune populations. Finally, we used sequence variation in the transcriptome data to determine host and donor chimerism at single-cell resolution from bone marrow mononuclear cells isolated from transplant patients.

Acoustic particle filter with adjustable effective pore size for automated sample preparation.

This article presents analysis and optimization of a microfluidic particle filter that uses acoustic radiation forces to remove particles larger than a selected size by adjusting the driving conditions of the piezoelectric transducer (PZT). Operationally, the acoustic filter concentrates microparticles to the center of the microchannel, minimizing undesirable particle adsorption to the microchannel walls. Finite element models predict the complex two-dimensional acoustic radiation force field perpendicular to the flow direction in microfluidic devices. We compare these results with experimental parametric studies including variations of the PZT driving frequencies and voltages as well as various particle sizes (0.5-5.0 microm in diameter). These results provide insight into the optimal operating conditions and show the efficacy of our device as a filter with an adjustable effective pore size. We demonstrate the separation of Saccharomyces cerevisiae from MS2 bacteriophage using our acoustic device. With optimized design of our microfluidic flow system, we achieved yields of greater than 90% for the MS2 with greater than 80% removal of the S. cerevisiae in this continuous-flow sample preparation device.

Microfluidic tools for biological sample preparation

Researchers at Lawrence Livermore National Laboratory are developing means to collect and identify fluid-based biological pathogens in the forms of proteins, viruses, and bacteria. To support detection instruments, we are developing a flexible fluidic sample preparation unit. The overall goal of this Microfluidic Module is to input a fluid sample, containing background particulates and potentially target compounds, and deliver a processed sample for detection. We are developing techniques for sample purification, mixing, and filtration that would be useful to many applications including immunologic and nucleic acid assays. Sample preparation functions are accomplished with acoustic radiation pressure, dielectrophoresis, and solid phase extraction. We are integrating these technologies into packaged systems with pumps and valves to control fluid flow and investigating small-scale detection methods.

DNA Purification of Biothreat Agents Using a Micropillar Chip

Processing large volume liquid samples for PCR-based infectious agent testing is ubiquitous among a wide variety of environmental samples. A number of different extraction protocols and devices are available to purify the nucleic acids from these complex samples. However, most of these approaches are optimized for specific kinds of samples and typically involve benchtop equipment and highly skilled personnel. Among the most common purification techniques are those that utilize a combination of chaotropic agents and random surfaces of glass (packed beds of micro-beads, fibers, particles, etc.) in a simple disposable plastic device. As an alternative surface, we have exploited the glass-surface properties of oxidized single crystal silicon in high-surface-area microstructures for silicon oxide-mediated nucleic acid extraction, purification, and concentration from large volume samples. One particular microstructure, a silicon pillar chip, provides highly-ordered and controlled surface interactions. The high aspect ratio (∼40) of the pillars provides an immense surface area within a relatively small volume, thus allowing for the capability to concentrate nucleic acids by several orders of magnitude. Here, we report on the effectiveness of this microfluidic chip in processing Francisella DNA in wastewater. The flow-through properties of the microfluidic chip enabled the entire procedure to be automated by embedding the chip in a reusable microfluidic cassette.Copyright

A Rapid, Flow-through, DNA Extraction Module for Integration into Microfluidic Systems

A reusable flow-through micropillar chip module for efficient, pathogen nucleic acid extraction, purification, and concentration was evaluated on large volume samples. The module was tested on aqueous samples of Francisella tularensis genomic DNA. Only 30 min were required to process a 75-ml sample, resulting in a 1000-fold concentration effect. The data demonstrates the potential of microfluidic approaches for flow-through processing, detection and genetic identification of pathogenic agents at low concentrations in real-world samples.