Background: In the study of transposable elements (TEs), the generation of a high confidence set of consensus sequences that represent the diversity of TEs found in a given genome is a key step in the path to investigate these fascinating genomic elements. Many algorithms and pipelines are available to automatically identify putative TE families present in a genome. Despite the availability of these valuable resources, producing a library of high-quality full-length TE consensus sequences largely remains a process of manual curation. This know-how is often passed on from mentor-to-mentee within research groups, making it difficult for those outside the field to access this highly specialised skill. Results: Our manuscript attempts to fill this gap by providing a set of detailed computer protocols, software recommendations and video tutorials for those aiming to manually curate TEs. Detailed step-by-step protocols, aimed at the complete beginner, are presented in the Supplementary Methods. Conclusions: The proposed set of programs and tools presented here will make the process of manual curation achievable and amenable to all researchers and in special to those new to the field of TEs.

Genome assemblies are currently being produced at an impressive rate by consortia and individual laboratories. The low costs and increasing efficiency of sequencing technologies now enable assembling genomes at unprecedented quality and contiguity. However, the difficulty to assemble repeat‐rich and GC‐rich regions (genomic “dark matter”) limits insights into the evolution of genome structure and regulatory networks. Here, we compare the efficiency of currently available sequencing technologies (short/linked/long reads and proximity ligation maps) and combinations thereof in assembling genomic dark matter. By adopting different de‐novo assembly strategies, we compare individual draft assemblies to a curated multiplatform reference assembly and identify the genomic features that cause gaps within each assembly. We show that a multiplatform assembly implementing long‐read, linked‐read, and proximity sequencing technologies performs best at recovering transposable elements, multi‐copy MHC genes, GC‐rich microchromosomes, and the repeat‐rich W chromosome. Telomere‐to‐telomere assemblies are not a reality yet for most organisms, but by leveraging technology choice it is now possible to minimize genome assembly gaps for downstream analysis. We provide a roadmap to tailor sequencing projects for optimized completeness of both the coding and non‐coding parts of non‐model genomes.

The genomics revolution has led to the sequencing of a large variety of nonmodel organisms often referred to as “whole” or “complete” genome assemblies. But how complete are these, really? Here, we use birds as an example for nonmodel vertebrates and find that, although suitable in principle for genomic studies, the current standard of short‐read assemblies misses a significant proportion of the expected genome size (7% to 42%; mean 20 ± 9%). In particular, regions with strongly deviating nucleotide composition (e.g., guanine‐cytosine‐[GC]‐rich) and regions highly enriched in repetitive DNA (e.g., transposable elements and satellite DNA) are usually underrepresented in assemblies. However, long‐read sequencing technologies successfully characterize many of these underrepresented GC‐rich or repeat‐rich regions in several bird genomes. For instance, only ~2% of the expected total base pairs are missing in the last chicken reference (galGal5). These assemblies still contain thousands of gaps (i.e., fragmented sequences) because some chromosomal structures (e.g., centromeres) likely contain arrays of repetitive DNA that are too long to bridge with currently available technologies. We discuss how to minimize the number of assembly gaps by combining the latest available technologies with complementary strengths. At last, we emphasize the importance of knowing the location, size and potential content of assembly gaps when making population genetic inferences about adjacent genomic regions.

The tuatara (Sphenodon punctatus)—the only living member of the reptilian order Rhynchocephalia (Sphenodontia), once widespread across Gondwana^1,2—is an iconic species that is endemic to New Zealand^2,3. A key link to the now-extinct stem reptiles (from which dinosaurs, modern reptiles, birds and mammals evolved), the tuatara provides key insights into the ancestral amniotes^2,4. Here we analyse the genome of the tuatara, which—at approximately 5 Gb—is among the largest of the vertebrate genomes yet assembled. Our analyses of this genome, along with comparisons with other vertebrate genomes, reinforce the uniqueness of the tuatara. Phylogenetic analyses indicate that the tuatara lineage diverged from that of snakes and lizards around 250 million years ago. This lineage also shows moderate rates of molecular evolution, with instances of punctuated evolution. Our genome sequence analysis identifies expansions of proteins, non-protein-coding RNA families and repeat elements, the latter of which show an amalgam of reptilian and mammalian features. The sequencing of the tuatara genome provides a valuable resource for deep comparative analyses of tetrapods, as well as for tuatara biology and conservation. Our study also provides important insights into both the technical challenges and the cultural obligations that are associated with genome sequencing.

Structural variation (SV) constitutes an important type of genetic mutations providing the raw material for evolution. Here, we uncover the genome-wide spectrum of intra- and interspecific SV segregating in natural populations of seven songbird species in the genus Corvus. Combining short-read (N = 127) and long-read re-sequencing (N = 31), as well as optical mapping (N = 16), we apply both assembly- and read mapping approaches to detect SV and characterize a total of 220,452 insertions, deletions and inversions. We exploit sampling across wide phylogenetic timescales to validate SV genotypes and assess the contribution of SV to evolutionary processes in an avian model of incipient speciation. We reveal an evolutionary young (~530,000 years) cis-acting 2.25-kb LTR retrotransposon insertion reducing expression of the NDP gene with consequences for premating isolation. Our results attest to the wealth and evolutionary significance of SV segregating in natural populations and highlight the need for reliable SV genotyping.