The Genome Sequencer (GS) FLX from 454 Life Sciences is the ultra high-throughput instrument for conducting the next generation of DNA sequencing. It potentially can generate one million sequencing readings with an average reading at about 250 bp within a single operation run. This technology has been used for de novo sequencing and assembly of whole genomes, re-sequencing whole genomes, and amplicon variation analysis. This tutorial will introduce the principle of 454 sequencing technology, potential applications, data type, and data analysis. The main focus will be on GS de novo Assembler, GS Mapper, and GS Amplicon Variation Analyzer.