Comparative Genome Annotation.

Newly sequenced genomes are being added to the tree of life at an unprecedented fast pace. A large proportion of such new genomes are phylogenetically close to previously sequenced and annotated genomes. In other cases, whole clades of closely related species or strains ought to be annotated simultaneously. Often, in subsequent studies, differences between the closely related species or strains are in the focus of research when the shared gene structures prevail. We here review methods for comparative structural genome annotation. The reviewed methods include classical approaches such as the alignment of protein sequences or protein profiles against the genome and comparative gene prediction methods that exploit a genome alignment to annotate either a single target genome or all input genomes simultaneously. We discuss how the methods depend on the phylogenetic placement of genomes, give advice on the choice of methods, and examine the consistency between gene structure annotations in an example. Furthermore, we provide practical advice on genome annotation in general.

Multi-Genome Annotation with AUGUSTUS.

Comparing multiple related genomes can help to improve their structural annotation. The accuracy and consistency of the predicted exon-intron structures of the protein coding genes can be higher when considering all genomes at once rather than annotating one genome at a time.The comparative gene prediction algorithm of AUGUSTUS performs such a multi-genome annotation. A multiple alignment of genomes is used to exploit evolutionary clues to conservation and negative selection. Further, AUGUSTUS exploits the fact that orthologous genes typically have congruent exon-intron structures. Comparative AUGUSTUS simultaneously predicts the genes in all input genomes. In this chapter we walk the reader through a small example from eight vertebrate species, including the construction of an alignment of the input genomes and how to integrate RNA-Seq evidence from multiple species for gene finding.

Sixteen diverse laboratory mouse reference genomes define strain-specific haplotypes and novel functional loci.

We report full-length draft de novo genome assemblies for 16 widely used inbred mouse strains and find extensive strain-specific haplotype variation. We identify and characterize 2,567 regions on the current mouse reference genome exhibiting the greatest sequence diversity. These regions are enriched for genes involved in pathogen defence and immunity and exhibit enrichment of transposable elements and signatures of recent retrotransposition events. Combinations of alleles and genes unique to an individual strain are commonly observed at these loci, reflecting distinct strain phenotypes. We used these genomes to improve the mouse reference genome, resulting in the completion of 10 new gene structures. Also, 62 new coding loci were added to the reference genome annotation. These genomes identified a large, previously unannotated, gene (Efcab3-like) encoding 5,874 amino acids. Mutant Efcab3-like mice display anomalies in multiple brain regions, suggesting a possible role for this gene in the regulation of brain development.

Comparative Annotation Toolkit (CAT)-simultaneous clade and personal genome annotation.

The recent introductions of low-cost, long-read, and read-cloud sequencing technologies coupled with intense efforts to develop efficient algorithms have made affordable, high-quality de novo sequence assembly a realistic proposition. The result is an explosion of new, ultracontiguous genome assemblies. To compare these genomes, we need robust methods for genome annotation. We describe the fully open source Comparative Annotation Toolkit (CAT), which provides a flexible way to simultaneously annotate entire clades and identify orthology relationships. We show that CAT can be used to improve annotations on the rat genome, annotate the great apes, annotate a diverse set of mammals, and annotate personal, diploid human genomes. We demonstrate the resulting discovery of novel genes, isoforms, and structural variants-even in genomes as well studied as rat and the great apes-and how these annotations improve cross-species RNA expression experiments.

Repeat associated mechanisms of genome evolution and function revealed by the Mus caroli and Mus pahari genomes.

Understanding the mechanisms driving lineage-specific evolution in both primates and rodents has been hindered by the lack of sister clades with a similar phylogenetic structure having high-quality genome assemblies. Here, we have created chromosome-level assemblies of the Mus caroli and Mus pahari genomes. Together with the Mus musculus and Rattus norvegicus genomes, this set of rodent genomes is similar in divergence times to the Hominidae (human-chimpanzee-gorilla-orangutan). By comparing the evolutionary dynamics between the Muridae and Hominidae, we identified punctate events of chromosome reshuffling that shaped the ancestral karyotype of Mus musculus and Mus caroli between 3 and 6 million yr ago, but that are absent in the Hominidae. Hominidae show between four- and sevenfold lower rates of nucleotide change and feature turnover in both neutral and functional sequences, suggesting an underlying coherence to the Muridae acceleration. Our system of matched, high-quality genome assemblies revealed how specific classes of repeats can play lineage-specific roles in related species. Recent LINE activity has remodeled protein-coding loci to a greater extent across the Muridae than the Hominidae, with functional consequences at the species level such as reproductive isolation. Furthermore, we charted a Muridae-specific retrotransposon expansion at unprecedented resolution, revealing how a single nucleotide mutation transformed a specific SINE element into an active CTCF binding site carrier specifically in Mus caroli, which resulted in thousands of novel, species-specific CTCF binding sites. Our results show that the comparison of matched phylogenetic sets of genomes will be an increasingly powerful strategy for understanding mammalian biology.