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Analysis of the satellitome of the flour beetle Tribolium freemani by high throughput sequencing

Satellite DNA (satDNA) are noncoding sequences located primarily in the heterochromatic regions of eukaryotic chromosomes. Due to their noncoding nature, they were often regarded as “junk” DNA, but recent studies have shown the important role of satDNA in chromosomal architecture and proper chromosome segregation [1]. The tandem organization of satellite monomers in eukaryotic genomes also presents a great technical challenge for researchers, and therefore satDNAs have often been omitted from the genome assemblies. However, due to the development of new sequencing technologies, research on satDNA has become more prevalent. The genome of the flour beetle Tribolium freemani is composed of huge heterochromatic blocks, which makes it a great model organism for studying satellite sequences. The main satDNA of the species T. freemani, making up 31% of the genome, has been described almost three decades ago. The existence of other satDNA in the genome of the T. freemani beetle has not yet been confirmed [2]. The goal of this study was to reveal the satellitome, the collection of satDNA in the T. freemani genome by using high-throughput sequencing of the second and third generation. Whole-genome sequencing was carried out using the Illumina and PacBio platforms. Short (151 bp) paired-end reads obtained by Illumina sequencing were analyzed by the bioinformatics tool TAREAN which is capable of identifying new satellites based on short unassembled reads [3]. Using PacBio sequencing we obtained ultra-long high- fidelity (HiFi) reads which were used to study the organization of the satellite monomers in the genome. By combining the aforementioned analyses, we defined 10 new low-copy satellites. Their portions in the genome vary between 0.013-0.24%, while their monomer sizes range from 63 to 266 bp. The tandem organization of these satDNA has been successfully proven by using the Southern blot method, and their chromosomal location was determined by fluorescence in situ hybridization. The new satellites are found dispersed in the genome, comprising arrays spanning more than 10 kilobases, but also show higher order repeat (HOR) structures. To conclude, by using the high-throughput sequencing, the T. freemani satellitome has been deciphered. Characterization of the previously unknown DNA sequences paves the way for a comprehensive understanding of the T. freemani genome as well as for further studies on the function of its hidden genome parts.

Satellitome ; Tribolium freemani ; WGS

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