The Genome Sequencer (GS) FLX from 454 Life Sciences is the high-throughput instrument for conducting nucleotide sequencing. It can potentially generate one million sequencing readings with an average reading at about 250 bp within a single operation run. This technology has been used for small genome sequencing, mass EST sequencing, and potentially large genome analysis. This tutorial will introduce the principle of 454 sequencing technology, data preparation, and data analysis, especially in sequence assembly and mapping. We will also discuss RNA and protein identification on the assembled genome.