Accurate and prompt gene structure annotation is a necessity for the utilization of genomic sequence data and large scale biological research. As the number of genomes being sequenced rapidly increases, there is a need for immediate gene structure annotation, which is distinct from the whole genome annotation that is conducted at the completion of sequencing. In completely sequenced genomes, inaccuracies in gene structure annotations can make subsequent analysis problematic. For instance, the incorrect assignment of exons to a gene can change the protein coded by the gene, which would have effects on the functional and evolutionary descriptions of the gene.;To address both of these needs, the Tracembler and yrGATE tools were developed. Tracembler provides the unique utility of sequence search, assembly and gene structure annotation upon the latest available fragmented sequence data. Discovery of microsynteny in soybean using adjacent Medicago truncatula genes as seeds was demonstrated using Tracembler. yrGATE ("your Gene Structure Annotation Tool for Eukaryotes") allows users to create their own gene structure annotations using high quality evidence, which can then be submitted to a community for review. yrGATE has been successfully used in re-annotating inaccurate published gene annotations and in annotating emerging genomes. Using both of these tools, on-demand gene annotation is a reality.;A thorough genome-wide identification of U12-type introns across seven organisms revealed that some U12-type introns did not have accurate corresponding gene structure annotations. In these cases, novel gene structure annotations were created. A subsequent investigation of orthologous genes with these introns yielded new discoveries. The first instances of U12-type intron conversion to U2-type and loss were recorded in orthologous genes among vertebrates and among plants. Since the divergence of plants and animals, U12-type introns were shown to have been primarily lost. Orthologs with conserved U12-type introns were found to experience reduced evolution in terms of gene structure, global protein sequence, and local protein sequence. A correlation between phase frequencies and exonic sequence conservation of these introns was described. The results of this investigation are presented through the CIWOG ("Common Introns Within Orthologous Genes") database to aid future research.