Abstract
Taxonomically restricted genes (TRGs) are unique for a defined group of organisms and may act as potential genetic determinants of lineage-specific, biological properties. Here, we explore the TRGs of highly diverse and economically important Bacillus bacteria by examining commonly used TRG identification parameters and data sources. We show the significant effects of sequence similarity thresholds, composition, and the size of the reference database in the identification process. Subsequently, we applied stringent TRG search parameters and expanded the identification procedure by incorporating an analysis of noncoding and non-syntenic regions of non-Bacillus genomes. A multiplex annotation procedure minimized the number of false-positive TRG predictions and showed nearly one-third of the alleged TRGs could be mapped to genes missed in genome annotations. We traced the putative origin of TRGs by identifying homologous, noncoding genomic regions in non-Bacillus species and detected sequence changes that could transform these regions into protein-coding genes. In addition, our analysis indicated that Bacillus TRGs represent a specific group of genes mostly showing intermediate sequence properties between genes that are conserved across multiple taxa and nonannotated peptides encoded by open reading frames.