Genome assembly, structural variants, and genetic differentiation between Lake Whitefish young species pairs (Coregonus sp.) with long and short reads

The parallel evolution of nascent pairs of ecologically differentiated species offers an opportunity to get a better glimpse at the genetic architecture of speciation. Of particular interest is our recent ability to consider a wider range of genomic variants, not only single-nucleotide polymorphisms (SNPs), thanks to long-read sequencing technology. We can now identify structural variants (SVs) like insertions, deletions, and other structural rearrangements, allowing further insights into the genetic architecture of speciation and how different variants are involved in species differentiation. Here, we investigated genomic patterns of differentiation between sympatric species pairs (Dwarf and Normal) belonging to the Lake Whitefish (Coregonus clupeaformis) species complex. We assembled the first reference genomes for both Dwarf and Normal Lake Whitefish, annotated the transposable elements, and analysed the genome in the light of related coregonid species. Next, we used a combination of long-read and short-read sequencing to characterize SVs and genotype them at population-scale using genome-graph approaches, showing that SVs cover five times more of the genome than SNPs. We then integrated both SNPs and SVs to investigate the genetic architecture of species differentiation in two different lakes and highlighted an excess of shared outliers of differentiation. In particular, a large fraction of SVs differentiating the two species was driven by transposable elements (TEs), suggesting that TE accumulation during a period of allopatry predating secondary contact may have been a key process in the speciation of the Dwarf and Normal Whitefish. Altogether, our results suggest that SVs play an important role in speciation and that by combining second and third generation sequencing we now have the ability to integrate SVs into speciation genomics.

ECCsplorer: a pipeline to detect extrachromosomal circular DNA (eccDNA) from next-generation sequencing data

Background Extrachromosomal circular DNAs (eccDNAs) are ring-like DNA structures physically separated from the chromosomes with 100 bp to several megabasepairs in size. Apart from carrying tandemly repeated DNA, eccDNAs may also harbor extra copies of genes or recently activated transposable elements. As eccDNAs occur in all eukaryotes investigated so far and likely play roles in stress, cancer, and aging, they have been prime targets in recent research—with their investigation limited by the scarcity of computational tools. Results Here, we present the ECCsplorer, a bioinformatics pipeline to detect eccDNAs in any kind of organism or tissue using next-generation sequencing techniques. Following Illumina-sequencing of amplified circular DNA (circSeq), the ECCsplorer enables an easy and automated discovery of eccDNA candidates. The data analysis encompasses two major procedures: first, read mapping to the reference genome allows the detection of informative read distributions including high coverage, discordant mapping, and split reads. Second, reference-free comparison of read clusters from amplified eccDNA against control sample data reveals specifically enriched DNA circles. Both software parts can be run separately or jointly, depending on the individual aim or data availability. To illustrate the wide applicability of our approach, we analyzed semi-artificial and published circSeq data from the model organisms Homo sapiens and Arabidopsis thaliana, and generated circSeq reads from the non-model crop plant Beta vulgaris. We clearly identified eccDNA candidates from all datasets, with and without reference genomes. The ECCsplorer pipeline specifically detected mitochondrial mini-circles and retrotransposon activation, showcasing the ECCsplorer’s sensitivity and specificity. Conclusion The ECCsplorer (available online at https://github.com/crimBubble/ECCsplorer) is a bioinformatics pipeline to detect eccDNAs in any kind of organism or tissue using next-generation sequencing data. The derived eccDNA targets are valuable for a wide range of downstream investigations—from analysis of cancer-related eccDNAs over organelle genomics to identification of active transposable elements.

Transposable elements contribute to the spatiotemporal microRNA landscape in human brain development

Transposable elements (TEs) contribute to the evolution of gene regulatory networks and are dynamically expressed throughout human brain development and disease. One gene regulatory mechanism influenced by TEs is the miRNA system of post-transcriptional control. miRNA sequences frequently overlap TE loci and this miRNA expression landscape is crucial for control of gene expression in adult brain and different cellular contexts. Despite this, a thorough investigation of the spatiotemporal expression of TE-embedded miRNAs in human brain development is lacking. Here, we identify a spatiotemporally dynamic TE-embedded miRNA expression landscape between childhood and adolescent stages of human brain development. These miRNAs sometimes arise from two apposed TEs of the same subfamily, such as for L2 or MIR elements, but in the majority of cases stem from solo TEs. They give rise to in silico predicted high-confidence pre-miRNA hairpin structures, likely represent functional miRNAs and have predicted genic targets associated with neurogenesis. TE-embedded miRNA expression is distinct in the cerebellum when compared to other brain regions, as has previously been described for gene and TE expression. Furthermore, we detect expression of previously non-annotated TE-embedded miRNAs throughout human brain development, suggestive of a previously undetected miRNA control network. Together, as with non-TE-embedded miRNAs, TE-embedded sequences give rise to spatiotemporally dynamic miRNA expression networks, the implications of which for human brain development constitute extensive avenues of future experimental research. To facilitate interactive exploration of these spatiotemporal miRNA expression dynamics, we provide the 'Brain miRTExplorer' web application freely accessible for the community.

The fifth Japanese meeting on biological function and evolution through interactions between hosts and transposable elements

The fifth Japanese meeting on host–transposon interactions, titled “Biological Function and Evolution through Interactions between Hosts and Transposable Elements (TEs),” was held online on August 26–27, 2021. The meeting was supported by National Institute of Genetics and aimed to bring together researchers studying the diverse roles of TEs in genome function and evolution, as well as host defense systems against TE mobility by chromatin and RNA modifications and protein-protein interactions. Here, we present the highlights of the talks.

The insertion and dysregulation of transposable elements in osteosarcoma and their association with patient event-free survival

The dysregulation of transposable elements (TEs) has been explored in a variety of cancers. However, TE activities in osteosarcoma (OS) have not been extensively studied yet. By integrative analysis of RNA-seq, whole-genome sequencing (WGS), and methylation data, we showed aberrant TE activities associated with dysregulations of TEs in OS tumors. Specifically, expression levels of LINE-1 and Alu of different evolutionary ages, as well as subfamilies of SVA and HERV-K, were significantly up-regulated in OS tumors, accompanied by enhanced DNA repair responses. We verified the characteristics of LINE-1 mediated TE insertions, including target site duplication (TSD) length (centered around 15 bp) and preferential insertions into intergenic and AT-rich regions as well as intronic regions of longer genes. By filtering polymorphic TE insertions reported in 1000 genome project (1KGP), besides 148 tumor-specific somatic TE insertions, we found most OS patient-specific TE insertions (3175 out of 3326) are germline insertions, which are associated with genes involved in neuronal processes or with transcription factors important for cancer development. In addition to 68 TE-affected cancer genes, we found recurrent germline TE insertions in 72 non-cancer genes with high frequencies among patients. We also found that +/− 500 bps flanking regions of transcription start sites (TSS) of LINE-1 (young) and Alu showed lower methylation levels in OS tumor samples than controls. Interestingly, by incorporating patient clinical data and focusing on TE activities in OS tumors, our data analysis suggested that higher TE insertions in OS tumors are associated with a longer event-free survival time.

Separable roles for RNAi in regulation of transposable elements and viability in the fission yeast Schizosaccharomyces japonicus

RNA interference (RNAi) is a conserved mechanism of small RNA-mediated genome regulation commonly involved in suppression of transposable elements (TEs) through both post-transcriptional silencing, and transcriptional repression via heterochromatin assembly. The fission yeast Schizosaccharomyces pombe has been extensively utilised as a model for studying RNAi pathways. However, this species is somewhat atypical in that TEs are not major targets of RNAi, and instead small RNAs correspond primarily to non-coding pericentromeric repeat sequences, reflecting a specialised role for the pathway in promoting heterochromatin assembly in these regions. In contrast, in the related fission yeast Schizosaccharomyces japonicus, sequenced small RNAs correspond primarily to TEs. This suggests there may be fundamental differences in the operation of RNAi pathways in these two related species. To investigate these differences, we probed RNAi function in S. japonicus. Unexpectedly, and in contrast to S. pombe, we found that RNAi is essential in this species. Moreover, viability of RNAi mutants can be rescued by mutations implicated in enhanching RNAi-independent heterochromatin propagation. These rescued strains retain heterochromatic marks on TE sequences, but exhibit derepression of TEs at the post-transcriptional level. Our findings indicate that S. japonicus retains the ancestral role of RNAi in facilitating suppression of TEs via both post-transcriptional silencing and heterochromatin assembly, with specifically the heterochromatin pathway being essential for viability, likely due to a function in genome maintenance. The specialised role of RNAi in heterochromatin assembly in S. pombe appears to be a derived state that emerged after the divergence of S. japonicus.

RNA-mediated nucleosome depletion is required for elimination of transposon-derived DNA.

Small RNAs are known to mediate silencing of transposable elements and other genomic loci, increasing nucleosome density and preventing undesirable gene expression. Post-zygotic development of the Paramecium somatic genome requires elimination of thousands of transposon remnants (IESs) and transposable elements that are scattered throughout the germline genome (Garnier et al. 2004). The elimination process is guided by Piwi-associated small RNAs and leads to precise cleavage at IES boundaries (Bouhouche et al. 2011; Furrer et al. 2017). Previous research suggests that small RNAs induce heterochromatin formation within IESs, which, in turn, is required for DNA elimination (Liu et al. 2007). Here we show that IES recognition and precise excision is facilitated by recruitment of a homolog of a chromatin remodeler ISWI, which depletes target genomic regions of nucleosomes, making the chromatin accessible for DNA cleavage. ISWI knockdown in Paramecium leads to pronounced inhibition of DNA elimination. Furthermore, nucleosome profiling indicates that ISWI is required for IES elimination in nucleosome-dense genomic regions, while other IESs do not require small RNAs or ISWI for excision. ISWI silencing notably also reduces DNA elimination precision, resulting in aberrant excision at alternative IES boundaries. In summary, we demonstrate that chromatin remodeling that increases DNA accessibility together with small RNAs are necessary for efficient and precise DNA elimination in Paramecium.