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.

Extrachromosomal Circular DNA (eccDNA): From Chaos to Function

Extrachromosomal circular DNA (eccDNA) is a type of double-stranded circular DNA that is derived and free from chromosomes. It has a strong heterogeneity in sequence, length, and origin and has been identified in both normal and cancer cells. Although many studies suggested its potential roles in various physiological and pathological procedures including aging, telomere and rDNA maintenance, drug resistance, and tumorigenesis, the functional relevance of eccDNA remains to be elucidated. Recently, due to technological advancements, accumulated evidence highlighted that eccDNA plays an important role in cancers by regulating the expression of oncogenes, chromosome accessibility, genome replication, immune response, and cellular communications. Here, we review the features, biogenesis, physiological functions, potential functions in cancer, and research methods of eccDNAs with a focus on some open problems in the field and provide a perspective on how eccDNAs evolve specific functions out of the chaos in cells.

The complete mitochondrial genomes of Notiophilus quadripunctatus Dejean, 1826 and Omophron limbatum (Fabricius, 1777): New insights into the mitogenome phylogeny of the Carabidae (Insecta, Coleoptera)

The Carabidae is by far the largest family of the Adephaga, with more than 40,000 described species. Whereas their phylogeny has been extensively studied, convergences and reversals in morphological traits prevent a robust phylogenetic concept so far. In this study, we sequenced the complete mitochondrial genomes of Notiophilus quadripunctatus (Nebriinae) and Omophrom limbatum (Omophroninae) using high-throughput sequencing. Both mitogenomes consisted of a single circular DNA molecule that encoded the typical 13 protein-coding genes, two subunits of mitochondrial RNAs, 22 tRNAs and a putative control region. Our phylogenetic study placed Omophrom limbatum as sister taxon to all other analyzed ground beetle species whereas Notiophilus quadripunctatus was identified as sister to Nebria brevicollis as part of the Nebriinae. The analyses also support the monophyly of the Cicindelidae but place Trachypachus holmbergi (Trachypachidae) within the Carabidae. Nevertheless, almost all carabid subfamilies with more than one analyzed species were identified as monophyla.

A variety of highly divergent eukaryotic ssDNA viruses in sera of pigs

Over the last decade, viral metagenomics has been established as a non-targeted approach for identifying viruses in stock animals, including pigs. This has led to the identification of a vast diversity of small circular ssDNA viruses. The present study focuses on the investigation of eukaryotic circular Rep-encoding single-stranded (CRESS) DNA viral genomes present in serum of commercially reared pigs from southern Brazil. Several CRESS DNA viral genomes were detected, including representatives of the families Smacoviridae (n=5), Genomoviridae (n=3), Redondoviridae (n=1), Nenyaviridae (n=1) and other yet unclassified genomes (n=9), plus a circular DNA molecule, which probably belongs to the phylum Cressdnaviricota. A novel genus within the family Smacoviridae, tentatively named ‘Suismacovirus’, comprising 21 potential new species, is proposed. Although the reported genomes were recovered from pigs with clinical signs of respiratory disease, further studies should examine their potential role as pathogens. Nonetheless, these findings highlight the diversity of circular ssDNA viruses in serum of domestic pigs, expand the knowledge on CRESS DNA viruses’ genetic diversity and distribution and contribute to the global picture of the virome of commercially reared pigs.

ICTV Virus Taxonomy Profile: Geminiviridae 2021

The family Geminiviridae includes viruses with mono- or bipartite single-stranded, circular DNA genomes of 2.5–5.2 kb. They cause economically important diseases in most tropical and subtropical regions of the world. Geminiviruses infect dicot and monocot plants and are transmitted by insect vectors. DNA satellites are associated with some geminiviruses. This is a summary of the International Committee on Taxonomy of Viruses (ICTV) Report on the family Geminiviridae which is available at ictv.global/report/geminiviridae.

Cell free extrachromosomal circular DNA is common in human urine

Cell free extrachromosomal circular DNA (eccDNA) is evolving as a potential biomarker in liquid biopsies for disease diagnosis. In this study, an optimized next generation sequencing-based Circle-Seq method was developed to investigate urinary cell free eccDNA (ucf-eccDNA) from 28 adult healthy volunteers (mean age = 28, 19 males/ 9 females). The genomic distributions and sequence compositions of ucf-eccDNAs were comprehensively characterized. Approximately 1.2 million unique ucf-eccDNAs are identified, covering 14.9% of the human genome. Comprehensive characterization of ucf-eccDNAs show that ucf-eccDNAs contain higher GC content than flanking genomic regions. Most eccDNAs are less than 1000 bp and present four pronounced peaks at 203, 361, 550 and 728 bp, indicating the association between eccDNAs and the numbers of intact nucleosomes. Analysis of genomic distribution of ucf-eccDNAs show that eccDNAs are found in all chromosomes but enriched in chromosomes i.e. chr.17, 19 and 20 with high density of protein-codding genes, CpG islands, SINE and simple repeat elements. Lastly, analysis of sequence motif signatures at eccDNA junction sites reveal that direct repeats (DRs) are commonly found, indicating a potential role of DRs in eccDNA biogenesis. This work underscores the deep sequencing analysis of ucf-eccDNAs and provides a valuable reference resource for exploring potential applications of ucf-eccDNA as diagnostic biomarkers of urogenital disorders in the future.Significance StatementExtrachromosomal circular DNA (eccDNA) is an important genetic element and a biomarker for disease diagnosis and treatment. In this study, we conduct a comprehensive characterization of urinary cell free eccDNA (ucf-eccDNA) in 28 heathy subjects. Over one million ucf-eccDNAs are identified. Ucf-eccDNAs are characterized as high GC content. The size of most ucf-eccDNAs is less than 1000 bp and enriched in four peaks resembling the size of single, double, triple, and quadruple nucleosomes. The genomic distribution of ucf-eccDNAs is enriched in generic regions, protein-coding genes, Alu, CpG islands, SINE and simple repeats. Sequence motif analysis of ucf-eccDNA junctions identified simple direct repeats (DRs) commonly presented in most eccDNAs, suggesting potential roles of DRs in eccDNA biogenesis.

Charakterystyka kolistych DNA u Eukarya

W komórkach eukariotycznych DNA występuje głównie w formie liniowej, upakowanej w chromosomy. Poza tym może też przybierać postać kolistych cząsteczek. Najdokładniej zbadane zostały koliste DNA odgrywające rolę genomów mitochondriów i chloroplastów. Niemniej zasób kolistych DNA u Eukarya jest znacznie szerszy. Obejmuje również pozachromosomowe cząsteczki (ang. extrachromosomal circular DNA; eccDNA): koliste formy rDNA, pierścienie telomerowe, małe polidyspersyjne DNA, mikroDNA oraz inne typy kolistych DNA o pochodzeniu jądrowym. Występowanie eccDNA potwierdzono u wszystkich organizmów testowanych w tym zakresie. Dotychczasowe badania wykazały, że niektóre eccDNA są obecne na każdym etapie cyklu komórkowego, podczas gdy inne pojawiają się i/lub ulegają nagromadzeniu w szczególnych okolicznościach. Dowiedziono, że akumulacja eccDNA nierzadko zachodzi w następstwie poważnej destabilizacji genomu, będącej wynikiem stanów chorobowych czy stresu. Choć w środowisku naukowym wzrasta zainteresowanie eccDNA, pozostają one słabo poznanym składnikiem genomów eukariotycznych. Nadal niewiele wiadomo na temat mechanizmów ich formowania, ewolucji oraz ich funkcji biologicznych.