Assembly and annotation of the mitochondrial minicircle genome of a differentiation-competent strain of Trypanosoma brucei

Kinetoplastids are protists defined by one of the most complex mitochondrial genomes in nature, the kinetoplast. In the sleeping sickness parasite Trypanosoma brucei, the kinetoplast is a chain mail-like network of two types of interlocked DNA molecules: a few dozen ~23-kb maxicircles, which are homologs of the mitochondrial genome of other eukaryotes, and thousands of ~1-kb minicircles. Maxicircles encode subunits of respiratory chain complexes and the mitoribosome. Several maxicircle-encoded mRNAs require extensive post-transcriptional RNA editing via addition or deletion of uridine residues for maturation. The process is mediated by hundreds of species of minicircle-encoded guide RNAs (gRNAs), but the precise number of minicircle classes and gRNA genes was unknown. Here we present the first essentially complete assembly and annotation of the kinetoplast genome of T. brucei. We have identified 391 minicircles, encoding not only ~930 predicted ‘canonical’ gRNA genes that cover nearly all known editing events, but also ~370 ‘non-canonical’ gRNAs genes of unknown function. Small RNA transcriptome data confirmed expression of the majority of both categories of gRNAs. Finally, we have used our data set to refine definitions for minicircle and gRNA structure, to explore dynamics of minicircle copy numbers, and to uncover novel features of these molecules.