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5S ribosomal DNAs (rDNAs) are arranged in tandem and are often under-represented in genome assemblies. In the present study, we performed a global and in-depth analysis of the 5S rDNAs in the model insect Tribolium castaneum and its closely related species Tribolium freemani. To accomplish this goal, we used our recently published genome assemblies based on Nanopore and PacBio long-read sequencing. Although these closely related species share the 5S rRNA gene sequence with high homology, they show a different organization of the 5S rDNA locus. Analysis of 5S rDNA arrays in T. castaneum revealed a typical tandemly repeated organization characterized by repeat units consisting of the 121 bp long 5S rRNA gene and the 71 bp long nontranscribed spacer (NTS). In contrast, T. freemani showed a much more complex organization of 5S rDNA arrays characterized by two patterns. The first is based on the association of 5S rRNA gene with arrays of a satellite DNA, representing the NTS sequence of the 5S rDNA genes in T. freemani. The second, more complex type is characterized by a somewhat less frequent occurrence of the 5S rRNA gene and its association with longer satellite DNA arrays that are regularly interrupted by Jockey-like retrotransposons. This organization, in which the ribosomal gene is associated with two completely different repetitive elements such as satellite DNAs and retrotransposons, suggests that the 5S rRNA gene, regardless of its crucial function in the genome, could be a subject of extremely dynamic genomic rearrangements.

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Introduction: The development of colorectal cancer (CRC) is a multistep process accompanied by the accumulation of mutations that start from specific precancerous lesion - colorectal adenomas (CA). CRC incidence and mortality can be reduced by the early identification of these neoplasm. Colonoscopy is the most widely used screening method for CRC identification. Nowadays, clinical research interest is shifting to the use of liquid biopsy that may help with the early diagnosis of CA and CRC. In our previous study, we identified long non-coding RNA MALAT1 gene amplification associated with the development of CA. Methods: This study aimed to describe the potential of MALAT1 expression levels in the adenoma tissue of patients used in the previous study by real-time qPCR. Furthermore, we analysed the plasma samples of an independent group of patients with CA (n=97), CRC (n=101), and cancer-free individuals (CFI, n=48). Results: There was no difference in the MALAT1 expression level between CA patients with or without MALAT1 amplification. However, the plasma MALAT1 expression levels were significantly upregulated in patients with CRC and CA compared to CFI (for both p<0.001). Moreover, a correlation between MALAT1 expression and histological types of adenomas was identified- high-CRC-risk adenomas also displayed the highest MALAT1 expression levels. Furthermore, in CRC patients, MALAT1 levels were associated with a response to therapy. Conclusion: MALAT1 expression levels could serve as a promising circulating biomarker for early CA and CRC diagnosis, and even as a predictor of therapy response in CRC patients.

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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".

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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.

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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.

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