de Yu

The next-generation sequencing technology and application

As one of the key technologies in biomedical research, DNA sequencing has not only improved its productivity with an exponential growth rate but also been applied to new areas of application over the past few years. This is largely due to the advent of newer generations of sequencing platforms, offering ever-faster and cheaper ways to analyze sequences. In our previous review, we looked into technical characteristics of the next-generation sequencers and provided prospective insights into their future development. In this article, we present a brief overview of the advantages and shortcomings of key commercially available platforms with a focus on their suitability for a broad range of applications.

The next-generation sequencing technology: A technology review and future perspective

As one of the most powerful tools in biomedical research, DNA sequencing not only has been improving its productivity in an exponential growth rate but also been evolving into a new layout of technological territories toward engineering and physical disciplines over the past three decades. In this technical review, we look into technical characteristics of the next-gen sequencers and provide prospective insights into their future development and applications. We envisage that some of the emerging platforms are capable of supporting the

Droplet-in-oil array for picoliter-scale analysis based on sequential inkjet printing

1000 genome and

A complete genome assembly of Glaciecola mesophila sp. nov. sequenced by using BIGIS-4 sequencer system

100 genome goals if given a few years for technical maturation. We also suggest that scientists from China should play an active role in this campaign that will have profound impact on both scientific research and societal healthcare systems.

A novel dual-well array chip for efficiently trapping single-cell in large isolated micro-well without complicated accessory equipment

In recent years, inkjet printing, as a new method to fabricate microdroplet microarrays, has been increasingly applied in the field of biochemical diagnostics. To further improve the general applicability of the inkjet printing technology in fabricating biochemical chips, in this work, we introduce a model to describe the multiple injection procedure implemented by the inkjet printing approach, with experimental verification. The multiple injection model demonstrates a new sequential inkjet printing method that generates picoliter-scale multicomponent droplet-in-oil arrays via multistep printing on uniform planar substrates. Based on our previous work on double-inkjet printing, this technique adapts the piezoelectric inkjet printing technology to fabricate an oil droplet array, into which multiple precise injections of secondary droplets with different compositions and volumes can be automatically printed in the required sequence, simultaneously addressing the evaporation issues associated with printing picoliter droplets without external assistance. In this paper, we first describe the theory and characterize the model, which account for the basic principles of sequential inkjet printing, as well as validate the design in terms of multiple injections, droplet fusion, and rapid mixing. The feasibility and effectiveness of the method are also demonstrated in a dual fluorescence assay and a β-galactosidase enzyme inhibition assay. We believe that applying the sequential inkjet printing methodology in existing inkjet printing devices will enhance their use as universal diagnostic tools as well as accelerate the adoption of inkjet printing in multistep screening experiments.

Improved picoliter-sized micro-reactors for high-throughput biological analysis

Using a pyrosequencing-based custom-made sequencer BIGIS-4, we sequenced a Gram-negative bacterium Glaciecola mesophila sp. nov. (Gmn) isolated from marine invertebrate specimens. We generated 152043 sequencing reads with a mean high-quality length of 406 bp, and assembled them using the BIGIS-4 post-processing module. No systematic low-quality data was detected beyond expected homopolymer-derived errors. The assembled Gmn genome is 5144318 bp in length and harbors 4303 annotated genes. A large number of metabolic genes correspond to various nutrients from surface marine invertebrates. Its abundant cold-tolerant and cellular signaling and related genes reveal a fundamental adaptation to low-temperature marine environment.

A novel picoliter droplet array for parallel real-time polymerase chain reaction based on double-inkjet printing

Conventional cell-sized well arrays have advantages of high occupancy, simple operation, and low cost for capturing single-cells. However, they have insufficient space for including reagents required for cell treatment or analysis, which restricts the wide application of cell-sized well arrays as a single-cell research tool alone. Here, we present a novel dual-well array chip, which integrates capture-wells (20u2009μm in diameter) with reaction-wells (100u2009μm in diameter) and describe a flow method for convenient single-cell analysis requiring neither complicated infra-structure nor high expenditure, while enabling highly efficient single cell trapping (75.8%) with only 11.3% multi-cells. Briefly, the cells are first loaded into the dual-wells by gravity and then multi-cells in the reaction-wells are washed out by phosphate buffer saline. Next, biochemical reagents are loaded into reaction-wells using the scraping method and the chip is packed as a sandwich structure. We thereby successfully measured intracellular β-galactosidase activity of K562 cells at the single-cell level. We also used computational simulations to illustrate the working principle of dual-well structure and found out a relationship between the wall shear stress distribution and the aspect ratio of the dual-well array chip which provides theoretical guidance for designing multi-wells chip for convenient single-cell analysis. Our work produced the first dual-well chip that can simultaneously provide a high occupancy rate for single cells and sufficient space for reagents, as well as being low in cost and simple to operate. We believe that the feasibility and convenience of our method will enhance its use as a practical single-cell research tool.

Reaction chamber for DNA sequenator

High-throughput pyrosequencing, carried out in millions of picoliter-sized reactors on a fiber-optic slide, is known for its longer read length. However, both optical crosstalk (which reduces the signal-to-noise ratio of CCD images) and chemical retention adversely affect the accuracy of chemiluminescence determination, and ultimately decrease the read length and the accuracy of pyrosequencing results. In this study, both titanium and oxidized aluminum films were deposited on the side walls and upper faces of micro-reactor slides to enhance optical isolation; the films reduced the inter-well crosstalk by one order of magnitude. Subsequently, chemical retention was shown to be caused by the lower diffusion coefficient of the side walls of the picoliter-sized reactors because of surface roughness and random pores. Optically isolated fiber-optic slides over-coated with silicon oxide showed smoother surface morphology, resulting in little chemical retention; this was further confirmed with theoretical calculations. Picoliter-sized micro-reactors coated with titanium-silicon oxide films showed the least inter-well optical crosstalk and chemical retention; these properties are expected to greatly improve the high-throughput pyrosequencing performance.