It was long thought that solely three different transposable elements - the I-element, the P-element and hobo - invaded natural D. melanogaster populations within the last century. By sequencing the ‘living fossils’ of Drosophila research, i.e. D. melanogaster strains sampled from natural populations at different time points, we show that a fourth TE, Tirant, invaded D. melanogaster populations during the past century. Tirant likely spread in D. melanogaster populations around 1938, followed by the I-element, hobo, and, lastly, the P-element. In addition to the recent insertions of the canonical Tirant, D. melanogaster strains harbor degraded Tirant sequences in the heterochromatin which are likely due to an ancient invasion, likely predating the split of D. melanogaster and D. simulans. These degraded insertions produce distinct piRNAs that were unable to prevent the novel Tirant invasion. In contrast to the I-element, P-element and hobo, we did not find that Tirant induces any hybrid dysgenesis symptoms. This absence of apparent phenotypic effects may explain the late discovery of the Tirant invasion. Recent Tirant insertions were found in all investigated natural populations. Populations from Tasmania carry distinct Tirant sequences, likely due to a founder effect. By investigating the TE composition of natural populations and strains sampled at different time points, insertion site polymorphisms, piRNAs and phenotypic effects, we provide a comprehensive study of a natural TE invasion.

In most animals, it is thought that the proliferation of a transposable element (TE) is stopped when the TE jumps into a piRNA cluster. Despite this central importance, little is known about the composition and the evolutionary dynamics of piRNA clusters. This is largely because piRNA clusters are notoriously difficult to assemble as they are frequently composed of highly repetitive DNA. With long reads, we may finally be able to obtain reliable assemblies of piRNA clusters. Unfortunately, it is unclear how to generate and identify the best assemblies, as many assembly strategies exist and standard quality metrics are ignorant of TEs. To address these problems, we introduce several novel quality metrics that assess: (a) the fraction of completely assembled piRNA clusters, (b) the quality of the assembled clusters and (c) whether an assembly captures the overall TE landscape of an organisms (i.e. the abundance, the number of SNPs and internal deletions of all TE families). The requirements for computing these metrics vary, ranging from annotations of piRNA clusters to consensus sequences of TEs and genomic sequencing data. Using these novel metrics, we evaluate the effect of assembly algorithm, polishing, read length, coverage, residual polymorphisms and finally identify strategies that yield reliable assemblies of piRNA clusters. Based on an optimized approach, we provide assemblies for the two Drosophila melanogaster strains Canton‐S and Pi2. About 80% of known piRNA clusters were assembled in both strains. Finally, we demonstrate the generality of our approach by extending our metrics to humans and Arabidopsis thaliana.

The advent of long-read sequencing holds great promise for research on transposable elements (TEs). Long reads may finally allow us to obtain reliable assemblies of repetitive regions, and thus shed light on many open questions in TE biology, such as the evolution of piRNA clusters, i.e., the master loci controlling TE activity. Currently, many different assembly strategies exist and it is not clear how to obtain the most suitable assemblies for TE research. In fact, it is not even clear how to best identify suitable assemblies as classic quality metrics such as BUSCO and NG50 are ignorant of TEs. To address these problems, we introduce four novel quality metrics that assess i) how well piRNA clusters are assembled (CUSCO) and ii) to which extent an assembly captures the TE landscape of an organism (TE abundance, SNPs and internal deletions). Using these novel metrics, we evaluate the effect of assemblers, polishing, read length, coverage, residual polymorphisms, and finally, identify suitable assembly strategies. Using an optimized approach, we provide high-quality assemblies for the two Drosophila melanogaster strains Canton-S and Pi2. Around 80% of the piRNA clusters were contiguously assembled in these two strains. Such high-quality assemblies will provide novel insights into the biology of TEs. It is, for example, an open question of whether piRNA clusters contain abundant presence/absence polymorphism of TE insertions, as expected when piRNA clusters are responsible for stopping TE invasions. A comparison of the sequences of our assembled piRNA clusters reveals that such polymorphisms are indeed abundantly found in clusters.

It was long thought that solely three different transposable elements -the I-element, the P-element and hobo -invaded natural D. melanogaster populations within the last century. By sequencing the 'living fossils' of Drosophila research, i.e. D. melanogaster strains sampled from natural populations at different time points, we show that a fourth TE, Tirant, invaded D. melanogaster populations during the past century. Tirant likely spread in D. melanogaster populations around 1938, followed by the I-element, hobo, and, lastly, the P-element. In addition to the recent insertions of the canonical Tirant, D. melanogaster strains harbour degraded Tirant sequences in the heterochromatin which are likely due to an ancient invasion, possibly predating the split of D. melanogaster and D. simulans. In contrast to the I-element, P-element and hobo, we did not find that Tirant induces any hybrid dysgenesis symptoms. This absence of apparent phenotypic effects may explain the late discovery of the Tirant invasion. Recent Tirant insertions were found in all investigated natural populations. Populations from Tasmania carry distinct Tirant sequences, likely due to a founder effect. By investigating the TE composition of natural populations and strains sampled at different time points, insertion site polymorphisms, piRNAs and phenotypic effects, we provide a comprehensive study of a natural TE invasion.