Using high-throughput sequencing to study genome rearrangement in Oxytricha

Abstract

Oxytricha trifallax is a ciliate, a single-celled eukaryote with two genomes. The germline genome is transcriptionally silent throughout most of the cell’s life, and after sexual conjugation 90-95% of the germline sequence is eliminated to form a streamlined somatic genome that will serve the cell’s transcriptional needs. This genome rearrangement process, which involves the deletion of hundreds of thousands of germline-limited sequences, is still poorly understood. In this thesis I use high-throughput sequencing to explore genome rearrangement from three different angles. In Chapter 2 I use RNA-seq to investigate lncRNA copies of somatic chromosomes, which Oxytricha produces during conjugation. I find that these “template” RNAs, which may help the cell arrange somatic segments in the correct order and orientation, are produced throughout development, with different species appearing at different times. This suggests that the cell may rearrange its nascent somatic chromosomes in a particular order, rather than simultaneously. In Chapter 3 I use long-read sequencing to update Oxytricha’s somatic genome assembly to a version where more than half of the chromosomes were sequenced without assembly. I find thousands of chromosome isoforms that would be masked by traditional assembly approaches, including some that involve variable recombination between multiple germline loci, a previously unknown source of variation in the somatic genome. Finally, in Chapter 4 I characterize the activity of TBE transposons in the Oxytricha germline. I find these transposons often insert near or within regions of somatic sequence. TBEs therefore continue to shape germline fragmentation. These results demonstrate the power of high-throughput sequencing to tackle complex, genome-scale problems in non-model systems.