Christopher P. Fredlake

Advantages and limitations of next-generation sequencing technologies: A comparison of electrophoresis and non-electrophoresis methods

The reference human genome provides an adequate basis for biological researchers to study the relationship between genotype and the associated phenotypes, but a large push is underway to sequence many more genomes to determine the role of various specificities among different individuals that control these relationships and to enable the use of human genome data for personalized and preventative healthcare. The current electrophoretic methodology for sequencing an entire mammalian genome, which includes standard molecular biology techniques for genomic sample preparation and the separation of DNA fragments using capillary array electrophoresis, remains far too expensive (

Ultrafast DNA sequencing on a microchip by a hybrid separation mechanism that gives 600 bases in 6.5 minutes

5 million) to make genome sequencing ubiquitous. The National Human Genome Research Institute has put forth goals to reduce the cost of human genome sequencing to

Polymer systems designed specifically for DNA sequencing by microchip electrophoresis: a comparison with commercially available materials.

100 000 in the short term and

Hydrophobically Modified Polyacrylamide Block Copolymers for Fast, High-Resolution DNA Sequencing in Microfluidic Chips

1000 in the long term to spur the innovative development of technologies that will permit the routine sequencing of human genomes for use as a diagnostic tool for disease. Since the announcement of these goals, several companies have developed and released new, non‐electrophoresis‐based sequencing instruments that enable massive throughput in the gathering of genomic information. In this review, we discuss the advantages and limitations of these new, massively parallel sequencers and compare them with the currently developing next generation of electrophoresis‐based genetic analysis platforms, specifically microchip electrophoresis devices, in the context of three distinct types of genetic analysis.

Divergent dispersion behavior of ssDNA fragments during microchip electrophoresis in pDMA and LPA entangled polymer networks.

To realize the immense potential of large-scale genomic sequencing after the completion of the second human genome (Venters), the costs for the complete sequencing of additional genomes must be dramatically reduced. Among the technologies being developed to reduce sequencing costs, microchip electrophoresis is the only new technology ready to produce the long reads most suitable for the de novo sequencing and assembly of large and complex genomes. Compared with the current paradigm of capillary electrophoresis, microchip systems promise to reduce sequencing costs dramatically by increasing throughput, reducing reagent consumption, and integrating the many steps of the sequencing pipeline onto a single platform. Although capillary-based systems require ≈70 min to deliver ≈650 bases of contiguous sequence, we report sequencing up to 600 bases in just 6.5 min by microchip electrophoresis with a unique polymer matrix/adsorbed polymer wall coating combination. This represents a two-thirds reduction in sequencing time over any previously published chip sequencing result, with comparable read length and sequence quality. We hypothesize that these ultrafast long reads on chips can be achieved because the combined polymer system engenders a recently discovered “hybrid” mechanism of DNA electromigration, in which DNA molecules alternate rapidly between reptating through the intact polymer network and disrupting network entanglements to drag polymers through the solution, similar to dsDNA dynamics we observe in single-molecule DNA imaging studies. Most importantly, these results reveal the surprisingly powerful ability of microchip electrophoresis to provide ultrafast Sanger sequencing, which will translate to increased system throughput and reduced costs.

DNA sequencing by microchip electrophoresis using mixtures of high- and low-molar mass poly(N,N-dimethylacrylamide) matrices.

Electrophoresis‐based DNA sequencing is the only proven technology for the de novo sequencing of large and complex genomes. Miniaturization of capillary array electrophoresis (CAE) instruments can increase sequencing throughput and decrease cost while maintaining the high quality and long read lengths that has made CAE so successful for de novo sequencing. The limited availability of high‐performance polymer matrices and wall coatings designed specifically for microchip‐sequencing platforms continues to be a major barrier to the successful development of a commercial microchip‐sequencing instrument. It has been generally assumed that the matrices and wall coatings that have been developed for use in commercial CAE instruments will be able to be implemented directly into microchip devices with little to no change in sequencing performance. Here, we show that sequencing matrices developed specifically for microchip electrophoresis systems can deliver read lengths that are 150–300 bases longer on chip than some of the most widely used polymer‐sequencing matrices available commercially. Additionally, we show that the coating ability of commercial matrices is much less effective in the borosilicate chips used in this study. These results lead to the conclusion that new materials must be developed to make high‐performance microfabricated DNA‐sequencing instruments a reality.

Thermophysical properties of imidazolium-based ionic liquids

By using a microfluidic electrophoresis platform to perform DNA sequencing, genomic information can be obtained more quickly and affordably than the currently employed capillary array electrophoresis instruments. Previous research in our group has shown that physically cross‐linked, hydrophobically modified polyacrylamide matrices separate dsDNA more effectively than linear polyacrylamide (LPA) solutions. Expanding upon this work, we have synthesized a series of LPA‐co‐dihexylacrylamide block copolymers specifically designed to electrophoretically sequence ssDNA quickly and efficiently on a microfluidic device. By incorporating very small amounts of N,N‐dihexylacrylamide, a hydrophobic monomer, these copolymer solutions achieved up to ∼10% increases in average DNA sequencing read length over LPA homopolymer solutions of matched molar mass. Additionally, the inclusion of the small amount of hydrophobe does not significantly increase the polymer solution viscosities, relative to LPA solutions, so that channel loading times between the copolymers and the homopolymers are similar. The resulting polymer solutions are capable of providing enhanced sequencing separations in a short period of time without compromising the ability to rapidly load and unload the matrix from a microfluidic device.

DNA sequencing and genotyping in miniaturized electrophoresis systems

Resolution of DNA fragments separated by electrophoresis in polymer solutions (“matrices”) is determined by both the spacing between peaks and the width of the peaks. Prior research on the development of high‐performance separation matrices has been focused primarily on optimizing DNA mobility and matrix selectivity, and gave less attention to peak broadening. Quantitative data are rare for peak broadening in systems in which high electric field strengths are used (>150 V/cm), which is surprising since capillary and microchip‐based systems commonly run at these field strengths. Here, we report results for a study of band broadening behavior for ssDNA fragments on a glass microfluidic chip, for electric field strengths up to 320 V/cm. We compare dispersion coefficients obtained in a poly(N,N‐dimethylacrylamide) (pDMA) separation matrix that was developed for chip‐based DNA sequencing with a commercially available linear polyacrylamide (LPA) matrix commonly used in capillaries. Much larger DNA dispersion coefficients were measured in the LPA matrix as compared to the pDMA matrix, and the dependence of dispersion coefficient on DNA size and electric field strength were found to differ quite starkly in the two matrices. These observations lead us to propose that DNA migration mechanisms differ substantially in our custom pDMA matrix compared to the commercially available LPA matrix. We discuss the implications of these results in terms of developing optimal matrices for specific separation (microchip or capillary) platforms.

What is the future of electrophoresis in large-scale genomic sequencing?

Previous studies have reported that mixed molar mass polymer matrices show enhanced DNA sequencing fragment separation compared with matrices formulated from a single average molar mass. Here, we describe a systematic study to investigate the effects of varying the amounts of two different average molar mass polymers on the DNA sequencing ability of poly(N,N‐dimethylacrylamide) (pDMA) sequencing matrices in microfluidic chips. Two polydisperse samples of pDMA, with weight‐average molar masses of 3.5 MDa and 770 kDa, were mixed at various fractional concentrations while maintaining the overall polymer concentration at 5% w/v. We show that although the separation of short DNA fragments depends strongly on the overall solution concentration of the polymer, inclusion of the high‐molar mass polymer is essential to achieve read lengths of interest (>400 bases) for many sequencing applications. Our results also show that one of the blended matrices, comprised of 3% 3.5 MDa pDMA and 2% 770 kDa pDMA, yields similar sequencing read lengths (>520 bases on average) to the high‐molar mass matrix alone, while also providing a fivefold reduction in zero‐shear viscosity. These results indicate that the long read lengths achieved in a viscous, high‐molar mass polymer matrix are also possible to achieve in a tuned, blended matrix of high‐ and low‐molar mass polymers with a much lower overall solution viscosity.