Ciliates have two different types of nuclei per cell, with one acting as a somatic, transcriptionally active nucleus (macronucleus; abbr. MAC) and another serving as a germline nucleus (micronucleus; abbr. MIC). Furthermore, Oxytricha trifallax undergoes extensive genome rearrangements during sexual conjugation and post-zygotic development of daughter cells. These rearrangements are necessary because the precursor MIC loci are often both fragmented and scrambled, with respect to the corresponding MAC loci. Such genome architectures are remarkably tolerant of encrypted MIC loci, because RNA-guided processes during MAC development reorganize the gene fragments in the correct order to resemble the parental MAC sequence. Here, we describe the germline organization of several nested and highly scrambled genes in Oxytricha trifallax. These include cases with multiple layers of nesting, plus highly interleaved or tangled precursor loci that appear to deviate from previously described patterns. We present mathematical methods to measure the degree of nesting between precursor MIC loci, and revisit a method for a mathematical description of scrambling. After applying these methods to the chromosome rearrangement maps of O. trifallax we describe cases of nested arrangements with up to five layers of embedded genes, as well as the most scrambled loci in O. trifallax.

Ciliates are microbial eukaryotes that undergo extensive programmed genome rearrangement, a natural genome editing process that converts long germline chromosomes into smaller gene-rich somatic chromosomes. Three well-studied ciliates include Oxytricha trifallax, Tetrahymena thermophila and Paramecium tetraurelia, but only the Oxytricha lineage has a massively scrambled genome, whose assembly during development requires hundreds of thousands of precise programmed DNA joining events, representing the most complex genome dynamics of any known organism. Here we study the emergence of such complex genomes by examining the origin and evolution of discontinuous and scrambled genes in the Oxytricha lineage. This study compares six genomes from three species, the germline and somatic genomes for Euplotes woodruffi, Tetmemena sp., and the model ciliate Oxytricha trifallax. To complement existing data, we sequenced, assembled and annotated the germline and somatic genomes of Euplotes woodruffi, which provides an outgroup, and the germline genome of Tetmemena sp.. We find that the germline genome of Tetmemena is as massively scrambled and interrupted as Oxytricha's : 13.6% of its gene loci require programmed translocations and/or inversions, with some genes requiring hundreds of precise gene editing events during development. This study revealed that the earlier-diverged spirotrich, E. woodruffi, also has a scrambled genome, but only roughly half as many loci (7.3%) are scrambled. Furthermore, its scrambled genes are less complex, together supporting the position of Euplotes as a possible evolutionary intermediate in this lineage, in the process of accumulating complex evolutionary genome rearrangements, all of which require extensive repair to assemble functional coding regions. Comparative analysis also reveals that scrambled loci are often associated with local duplications, supporting a gradual model for the origin of complex, scrambled genomes via many small events of DNA duplication and decay.

Ciliates have two different types of nuclei per cell, with one acting as a somatic, transcriptionally active nucleus (macronucleus; abbr. MAC) and another serving as a germline nucleus (micronucleus; abbr. MIC). Furthermore, Oxytricha trifallax undergoes extensive genome rearrangements during sexual conjugation and post-zygotic development of daughter cells. These rearrangements are necessary because the precursor MIC loci are often both fragmented and scrambled, with respect to the corresponding MAC loci. Such genome architectures are remarkably tolerant of encrypted MIC loci, because RNA-guided processes during MAC development reorganize the gene fragments in the correct order to resemble the parental MAC sequence.Here, we describe the germline organization of several nested and highly scrambled genes in Oxytricha trifallax. These include cases with multiple layers of nesting, plus highly interleaved or tangled precursor loci that appear to deviate from previously described patterns. We present mathematical methods to measure the degree of nesting between precursor MIC loci, and revisit a method for a mathematical description of scrambling. After applying these methods to the chromosome rearrangement maps of O. trifallax we describe cases of nested arrangements with up to five layers of embedded genes, as well as the most scrambled loci in O. trifallax.

DNA rearrangements are important in various contexts, such as in vertebrate immunity, and cancer genome instability. The single-celled eukaryoteOxytricha trifallaxundergoes massive and reproducible genome rearrangement during post-zygotic development, making it a compelling model organism to study DNA rearrangements. To date, computational tools for the extraction and analysis of rearrangement annotations lack transparency and rely on assumptions that may not hold for all analyzed data, leading to irreproducibility of results and loss of information through data filtering or misrepresentation. An implementation of a procedure for the annotation and analysis of DNA rearrangement as a web application is discussed and tested. The resulting annotations provide an improvement over previous annotations in the following manner. (a) SDRAP achieves more complete precursor-product mappings than previous software (b) the software allows for full transparency of all parameters used during the annotation and therefore facilitates reproducible results, and (c) this parameter transparency makes SDRAP suitable for comparison of genomic data from different sources, including cancer genomes. This work introduces a theoretical framework and software to systematically extract and analyze annotations from pairs of genome assemblies corresponding to precursor and product rearrangement data. The software makes no assumptions about the structure of the rearrangements, and permits the user to select parameters to suit the data.