Alternative techniques for DNA sequencing can be grouped into several categories including microelectrophoretic methods, sequencing by hybridization, real-time observation of single molecules and cyclic-array sequencing. Preparation is accomplished by random fragmentation of DNA, followed by in vitro ligation of common adaptor sequences. The generation of clonal clustered amplicons to serve as sequencing characteristic can be achieved by in situ polonies, emulsion PCR or bridge PCR. PCR amplicons derived from any given single molecule end up spatially clustered, either to a single location on a planar substrate, or to the surface of micron-scale beads. The sequencing process consists of sequential cycles of enzyme-driven biochemistry and imaging-based data collection. Data are obtained by imaging of the full array at each cycle. Advantages of second-generation or cyclic-array strategies, include in vitro construction of a sequencing library, followed by in vitro clonal amplification to generate sequencing features. 7
Array-based sequencing allows for a higher degree of affinity than conventional capillary-based sequencing. Since array features are immobilized to a planar surface, they can be enzymatically influenced by a single reagent volume. The reasonable reagent volume per feature is on the scale of pico liters or femtoliters. Differences between second generation sequencing and the Sanger method result in lower costs for DNA sequence production. Some disadvantages of the second generation sequencing techniques include shorter read-lengths and decreased raw accuracy. Although these technologies will continue to improve and progress concerning these parameters. 7
The basic idea of sequencing by hybridization identifies variant positions using differential hybridization of labeled nucleic acid fragments to an array of oligonucleotide probes. With the approach taken by Affymetrix and Perlegen in performing large-scale SNP discovery in human, mouse and yeast, each potential single-base substitution is portrayed on the array by an unrelated feature. Roche NimbleGen takes a two-tier approach, first with an array directed at accomplishing estimated localization, and a second custom array directed at pinpointing and verification of variant positions, in performing sequencing by hybridization of microbial genomes. Microarrays are practical and cost effective for genomic re-sequencing as well as other genome-scale applications, but as next-generation sequencing technologies begin to compete it is unclear what will happen. 7
Limitations of microarrays include sequences that are recurring or susceptible to cross-hybridization cannot be examined easily, how de novo sequencing can be accomplished with hybridization-based strategies remains unclear; and false positives present a problem without careful data analysis, and is unclear how to acquire the equivalent of redundant coverage that is achievable with cyclic-array sequencing. Although sequencing...