Trends in Biotechnology
ReviewToward nanoscale genome sequencing
Introduction
The ability to read DNA, also known as sequencing, is critical to understanding inheritance, individuality, disease and evolution. The ability to sequence genomes faster and more cheaply than is possible today promises to accelerate our understanding of the genetic origin of diseases. The potential outcomes of such improvements include: the development of personalized medicines, the rapid analysis of genotypes and haplotypes, the identification of pathogens (e.g. the development of bio-warfare sensors), the discovery of cell-lineage patterns, and the exploration of microbial genomes for agricultural, environmental and therapeutic goals.
The state-of-the-art technology in human genome sequencing uses Sanger sequencing (Box 1) and is capable of sequencing 89% of the DNA in a typical run; the length of an individual read is 805 Q20 bases (sequencing bases with an error rate < 1%) [1]. A high-throughput genome center typically performs 106 reactions every month; this throughput and corresponding read length approximates a mammalian-sized genome. To ensure accuracy and contiguity in the sequencing method, redundancy equivalent to six times the read length of the genome is required. The total expenditure for sequencing a typical mammalian-sized genome is six months in time and costs approximately US$12 million.
This review describes recent trends in scaling traditional genome sequencing technologies to the microscale, as well as the introduction of nanoscale methods that could be used to sequence genomes. This review does not detail the significant advances in recombinant DNA engineering [2], fluorescent dye development [3], capillary electrophoresis [4], automation 5, 6 and computation methods [7] that provide the infrastructure to quantify and interpret rapidly the data produced from a sequencing reaction.
Section snippets
Miniaturization of existing genome sequencing technology
The method for sequencing DNA developed by Sanger et al. as well as its variations were crucial in obtaining the complete map of a human genome 8, 9. Recent advances in Sanger sequencing include the development of alternative methods to excite fluorescent dyes (e.g. pulsed multi-line excitation [10]) and new strategies for fluorescence detection (e.g. using the lifetime of fluorescence as the ‘signal’ from the dye [11]). Microfabricated devices that perform Sanger sequencing might reduce the
Advances in genome sequencing at the nanoscale
Optical trapping of two polystyrene beads, where one bead is attached to an RNA polymerase and the other bead is attached to a short strand of template DNA, can be used to sequence DNA [23]. This single-molecule method can detect the processive motion of the polymerase as it incorporates nucleotides along the template DNA. When the concentration of one type of nucleotides is limited, the polymerase stops on the template DNA at the position that precedes a low-abundance nucleotide; the entire
Toward the US$1000-genome
The performance of established and emerging DNA sequencing methods are quantified in Table 1 according to cost, speed, accuracy and read length. In addition, the simulated performance of a ‘US$1000-genome sequencing technology’ is also estimated. The concept of a US$1000-genome sequencing technology is an idealized and ambitious objective, which aims to develop instruments that allow for the sequencing of DNA relying on cheaper and faster methods than those currently in use, while maintaining
Conclusion
Conventional Sanger sequencing remains the method of choice for genome analysis. However, the miniaturization of this method using microfluidics and microelectrophoresis can present significant cost reductions (on a per read basis). It is clear that the serial loading of microchannels for sample purification will limit the application of miniaturized Sanger sequencing technologies, and the Massively Parallel Strategies that exploit clonal amplification might prevail in future applications.
Acknowledgements
The authors gratefully acknowledge financial support from the Gordon and Betty Moore Foundation and the National Academies Keck Future Initiative.
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