Casey Lamers

High-resolution human genome structure by single-molecule analysis

Variation in genome structure is an important source of human genetic polymorphism: It affects a large proportion of the genome and has a variety of phenotypic consequences relevant to health and disease. In spite of this, human genome structure variation is incompletely characterized due to a lack of approaches for discovering a broad range of structural variants in a global, comprehensive fashion. We addressed this gap with Optical Mapping, a high-throughput, high-resolution single-molecule system for studying genome structure. We used Optical Mapping to create genome-wide restriction maps of a complete hydatidiform mole and three lymphoblast-derived cell lines, and we validated the approach by demonstrating a strong concordance with existing methods. We also describe thousands of new variants with sizes ranging from kb to Mb.

Rapid Prototyping of Arrayed Microfluidic Systems in Polystyrene for Cell-Based Assays

Microfluidic cell-based systems have enabled the study of cellular phenomena with improved spatiotemporal control of the microenvironment and at increased throughput. While poly(dimethylsiloxane) (PDMS) has emerged as the most popular material in microfluidics research, it has specific limitations that prevent microfluidic platforms from achieving their full potential. We present here a complete process, ranging from mold design to embossing and bonding, that describes the fabrication of polystyrene (PS) microfluidic devices with similar cost and time expenditures as PDMS-based devices. Emphasis was placed on creating methods that can compete with PDMS fabrication methods in terms of robustness, complexity, and time requirements. To achieve this goal, several improvements were made to remove critical bottlenecks in existing PS embossing methods. First, traditional lithographic techniques were adapted to fabricate bulk epoxy molds capable of resisting high temperatures and pressures. Second, a method was developed to emboss through-holes in a PS layer, enabling creation of large arrays of independent microfluidic systems on a single device without need to manually create access ports. Third, thermal bonding of PS layers was optimized in order to achieve quality bonding over large arrays of microsystems. The choice of materials and methods was validated for biological function in two different cell-based applications to demonstrate the versatility of our streamlined fabrication process.

Single-Molecule Approach to Bacterial Genomic Comparisons via Optical Mapping

Modern comparative genomics has been established, in part, by the sequencing and annotation of a broad range of microbial species. To gain further insights, new sequencing efforts are now dealing with the variety of strains or isolates that gives a species definition and range; however, this number vastly outstrips our ability to sequence them. Given the availability of a large number of microbial species, new whole genome approaches must be developed to fully leverage this information at the level of strain diversity that maximize discovery. Here, we describe how optical mapping, a single-molecule system, was used to identify and annotate chromosomal alterations between bacterial strains represented by several species. Since whole-genome optical maps are ordered restriction maps, sequenced strains of Shigella flexneri serotype 2a (2457T and 301), Yersinia pestis (CO 92 and KIM), and Escherichia coli were aligned as maps to identify regions of homology and to further characterize them as possible insertions, deletions, inversions, or translocations. Importantly, an unsequenced Shigella flexneri strain (serotype Y strain AMC[328Y]) was optically mapped and aligned with two sequenced ones to reveal one novel locus implicated in serotype conversion and several other loci containing insertion sequence elements or phage-related gene insertions. Our results suggest that genomic rearrangements and chromosomal breakpoints are readily identified and annotated against a prototypic sequenced strain by using the tools of optical mapping.

A Whole-Genome Shotgun Optical Map of Yersinia pestis Strain KIM

ABSTRACT Yersinia pestis is the causative agent of the bubonic, septicemic, and pneumonic plagues (also known as black death) and has been responsible for recurrent devastating pandemics throughout history. To further understand this virulent bacterium and to accelerate an ongoing sequencing project, two whole-genome restriction maps (XhoI and PvuII) of Y. pestis strain KIM were constructed using shotgun optical mapping. This approach constructs ordered restriction maps from randomly sheared individual DNA molecules directly extracted from cells. The two maps served different purposes; the XhoI map facilitated sequence assembly by providing a scaffold for high-resolution alignment, while the PvuII map verified genome sequence assembly. Our results show that such maps facilitated the closure of sequence gaps and, most importantly, provided a purely independent means for sequence validation. Given the recent advancements to the optical mapping system, increased resolution and throughput are enabling such maps to guide sequence assembly at a very early stage of a microbial sequencing project.

An Automated High-Content Assay for Tumor Cell Migration through 3-Dimensional Matrices

High-content tumor cell migration assays in 3-dimensional (3D) extracellular matrix are a powerful tool for modeling and understanding the biology of this critical step in the process of metastasis. Currently available methods offer very limited throughput and are not amenable to studies of comparative pharmacology or small-scale screening. The authors present an automated approach to high-content tumor cell migration assays. A standard screening-sized plate with an array of microchannels was designed and constructed from common thermoplastics. After filling the channels with 3D matrix, cells were placed at one end of the channel, and migration into the channel was monitored via an imaging system. All liquid-handling steps were performed by standard liquid-handling robotics. Tumor cell migration in the channel was truly 3D and correlated with metastatic potential. The information-rich data from these assays were used to rank the potency of compounds inhibiting migration through 3D collagen as well as to gain additional insights into the compounds’ activities related to cell health. This approach is compatible with a variety of multiparametric, morphological, and/or kinetic readouts.

Image-based analysis of primary human neutrophil chemotaxis in an automated direct-viewing assay.

Multi-well assays based on the Boyden chamber have enabled highly parallel studies of chemotaxis-the directional migration of cells in response to molecular gradients-while direct-viewing approaches have allowed more detailed questions to be asked at low throughput. Boyden-based plates provide a count of cells that pass through a membrane, but no information about cell appearance. In contrast, direct-viewing devices enable the observation of cells during chemotaxis, which allows measurement of many parameters including area, shape, and location. Here we show automated chemotaxis and cell morphology assays in a 96-unit direct-viewing plate. Using only 12000 primary human neutrophils per datum, we measured dose-dependent stimulation and inhibition of chemotaxis and quantified the effects of inhibitors on cell area and elongation. With 60 parallel conditions we demonstrated 5-fold increase in throughput compared to previously reported direct-viewing approaches.