Applications of ultra-high-throughput sequencing.

Abstract

The genomics era has enabled scientists to more readily pose truly global questions regarding mutation, evolution, gene and genome structure, function, and regulation. Just as Sanger sequencing ushered in a paradigm shift that enabled the molecular basis of biological questions to be directly addressed, to an even greater degree, ultra-high-throughput DNA sequencing is poised to dramatically change the nature of biological research. New sequencing technologies have opened the door for novel questions to be addressed at the level of the entire genome in the areas of comparative genomics, systems biology, metagenomics, and genome biology. These new sequencing technologies provide a tremendous amount of DNA sequence data to be collected at an astounding pace, with reduced costs, effort, and time as compared to Sanger sequencing. Applications of ultra-high-throughput sequencing (UHTS) are essentially limited only by the imaginations of researchers, and include genome sequencing/resequencing, small RNA discovery, deep SNP discovery, chromatin immunoprecipitation (ChIP) and RNA immunoprecipitation (RIP) coupled with sequence identification, transcriptome analysis including empirical annotation, discovery and characterization of alternative splicing, and gene expression profiling. This technology will have a profound impact on plant breeding, biotechnology, and our fundamental understanding of plant evolution, development, and environmental responses. In this chapter, we provide an overview of UHTS approaches and their applications. We also describe a protocol we have developed for deep sequencing of plant transcriptomes using the Illumina/Solexa sequencing platform.