The red flour beetle Tribolium castaneum is an important pest of stored agricultural products and the first beetle whose genome was sequenced. So far, one high-copy-number and ten moderate-copy-number satellite DNAs (satDNAs) have been described in the assembled part of its genome. In this work, we aimed to catalog the entire collection of T. castaneum satDNAs. We resequenced the genome using Illumina technology and predicted potential satDNAs via graph-based sequence clustering. In this way, we discovered 46 novel satDNAs that occupied a total of 2.1% of the genome and were, therefore, considered low-copy-number satellites. Their repeat units, preferentially 140–180 bp and 300–340 bp long, showed a high A + T composition ranging from 59.2 to 80.1%. In the current assembly, we annotated the majority of the low-copy-number satDNAs on one or a few chromosomes, discovering mainly transposable elements in their vicinity. The current assembly also revealed that many of the in silico predicted satDNAs were organized into short arrays not much longer than five consecutive repeats, and some of them also had numerous repeat units scattered throughout the genome. Although 20% of the unassembled genome sequence masked the genuine state, the predominance of scattered repeats for some low-copy satDNAs raises the question of whether these are essentially interspersed repeats that occur in tandem only sporadically, with the potential to be satDNA “seeds”.

Eukaryotic genomes are replete with satellite DNAs (satDNAs), large stretches of tandemly repeated sequences that are mostly underrepresented in genome assemblies. Here we combined nanopore long-read sequencing with a reference-guided assembly approach to generate an improved, high-quality genome assembly, TcasONT, of the model beetleTribolium castaneum. Enriched by 45 Mb in repetitive regions, the new assembly comprises almost the entire genome sequence. We use the enhanced assembly to conduct global and in-depth analyses of abundant euchromatic satDNAs. Unexpectedly, we show the extensive spread of satDNAs in gene-rich regions, including long arrays. The sequence similarity relationships between satDNA monomers and arrays indicate a recent exchange of satDNA arrays between different chromosomes. We propose a scenario of their genome dynamics characterized by repeated bursts of satDNAs spreading through euchromatin, followed by a process of elongation and homogenization of arrays. We find that suppressed recombination on the X Chromosome has no significant effect on the spread of satDNAs but the X rather tolerates the amplification of satDNAs into longer arrays. Analyses of arrays’ neighboring regions show a tendency of one satDNA to be associated with transposable-like elements. Using 2D electrophoresis followed by Southern blotting, we prove Cast satDNAs’ presence in the fraction of extrachromosomal circular DNA (eccDNA). We point to two mechanisms that enable this satDNA spread to occur: transposition by transposable elements and insertion mediated by eccDNA. The presence of such a large proportion of satDNA in gene-rich regions inevitably gives rise to speculation about their possible influence on gene expression.

The long-read Nanopore sequencing has been recently applied for assembly of complex genomes and analysis of linear genome organization. The most critical factor for successful long-read sequencing is extraction of high molecular weight (HMW) DNA of sufficient purity and quantity. The challenges associated with input DNA quality are further amplified when working with extremely small insects with hard exoskeletons. Here, we optimized the isolation of HMW DNA from the model beetle Tribolium and tested for use in Nanopore sequencing. We succeeded in overcoming all the difficulties in HMW handling and library preparation that were encountered when using published protocols and commercial kits. Isolation of nuclei and subsequent purification of DNA on an anion-exchange chromatography column resulted in genomic HMW DNA that was efficiently relaxed, of optimal quality and in sufficient quantity for Nanopore MinION sequencing. DNA shearing increased average N50 read values up to 26 kb and allowed us to use a single flow cell in multiple library loads for a total output of more than 13 Gb. Although our focus was on T. castaneum and closely related species, we expect that this protocol, with appropriate modifications, could be extended to other insects, particularly beetles.

Satellite DNAs (satDNAs) are tandemly repeated sequences that make up a significant portion of almost all eukaryotic genomes. Although satDNAs have been shown to play an important role in genome organization and evolution, they are relatively poorly analyzed, even in model organisms. One of the main reasons for the current lack of in-depth studies on satDNAs is their underrepresentation in genome assemblies. Due to complexity, abundance, and highly repetitive nature of satDNAs, their analysis is challenging, requiring efficient tools that ensure accurate annotation and comprehensive genome-wide analysis. We present a novel pipeline, named satellite DNA Exploration (SatXplor), designed to robustly characterize satDNA elements and analyze their arrays and flanking regions. SatXplor is benchmarked against other tools and curated satDNA datasets from diverse species, including mice and humans, showcase its versatility across genomes with varying complexities and satDNA profiles. Component algorithms excel in the identification of tandemly repeated sequences and, for the first time, enable evaluation of satDNA variation and array annotation with the addition of information about surrounding genomic landscape. SatXplor is an innovative pipeline for satDNA analysis that can be paired with any tool used for satDNA detection, offering insights into the structural characteristics, array determination, and genomic context of satDNA elements. By integrating various computational techniques, from sequence analysis and homology investigation to advanced clustering and graph-based methods, it provides a versatile and comprehensive approach to explore the complexity of satDNA organization and understand the underlying mechanisms and evolutionary aspects. It is open-source and freely accessible at https://github.com/mvolar/SatXplor.

The flour beetle Tribolium freemani is a sibling species of the model organism and important pest Tribolium castaneum. The two species are so closely related that they can produce hybrid progeny, but the genetic basis of their differences has not been revealed. In this work, we sequenced the T. freemani genome by applying PacBio HiFi technology. Using the well-assembled T. castaneum genome as a reference, we assembled 262 Mb of the T. freemani genomic sequence and anchored it in 10 linkage groups corresponding to nine autosomes and sex chromosome X. The assembly showed 99.8% completeness of conserved insect genes, indicating a high-quality reference genome. Comparison with the T. castaneum assembly revealed that the main differences in genomic sequence between the two sibling species come from repetitive DNA, including interspersed and tandem repeats. In this work, we also provided the complete assembled mitochondrial genome of T. freemani. Although the genome assembly needs to be ameliorated in tandemly repeated regions, the first version of the T. freemani reference genome and the complete mitogenome presented here represent useful resources for comparative evolutionary studies of related species and for further basic and applied research on different biological aspects of economically important pests.

Eukaryotic genomes are replete with satellite DNAs (satDNAs), large stretches of tandemly repeated sequences which are mostly underrepresented in genome assemblies. Here we combined Nanopore long-read sequencing with a reference-guided assembly approach, to generate an improved, high-quality genome assembly TcasONT of the model beetle Tribolium castaneum . Enriched by 45 Mb in the repetitive part, the new assembly comprises almost the entire genome sequence. We used the enhanced assembly to conduct global and in-depth analyses of abundant euchromatic satDNAs, Cast1-Cast9. Contrary to the commonly adopted view, our finding showed the extensive spread of satDNAs in gene-rich regions, including long arrays. The results of the principal component analysis of monomers and sequence similarity relationships between satDNA arrays, revealed an occurrence of recent satDNAs array exchange between different chromosomes. We proposed a scenario of their genome dynamics characterized by repeated bursts of satDNAs spreading through euchromatin, followed by a process of elongation and homogenization of arrays. We also found that suppressed recombination on the X chromosome has no significant effect on the spread of satDNAs, but rather tolerates the amplification of these satDNAs into longer arrays. Analyses of arrays’ neighboring regions showed a tendency of one Cast satDNA to be associated with transposon-like elements. Using 2D electrophoresis followed by Southern blotting, we proved Cast satDNAs presence in the fraction of extrachromosomal circular DNA (eccDNA). We point to two mechanisms that enable the said satDNA spread to occur: transposition by transposable elements and insertion mediated by eccDNA. The presence of such a large proportion of satDNA in gene-rich regions inevitably gives rise to speculation about their possible influence on gene expression.