Amplicon-Based Next Generation Sequencing for Rapid Identification of Rickettsia and Ectoparasite Species from Entomological Surveillance in Thailand

Background: Next generation sequencing (NGS) technology has been used for a wide range of epidemiological and surveillance studies. Here, we used amplicon-based NGS to species identify Rickettsia and their arthropod hosts from entomological surveillance. Methods: During 2015–2016, we screened 1825 samples of rodents and ectoparasites collected from rodents and domestic mammals (dog, cat, and cattle) across Thailand for Rickettsia. The citrate synthase gene was amplified to identify Rickettsia to species, while the Cytochrome Oxidase subunit I (COI) and subunit II (COII) genes were used as target genes for ectoparasite identification. All target gene amplicons were pooled for library preparation and sequenced with Illumina MiSeq platform. Result: The highest percentage of Rickettsia DNA was observed in fleas collected from domestic animals (56%) predominantly dogs. Only a few samples of ticks from domestic animals, rodent fleas, and rodent tissue were positive for Rickettisia DNA. NGS based characterization of Rickettsia by host identified Rickettsia asembonensis as the most common bacteria in positive fleas collected from dogs (83.2%) while “Candidatus Rickettsia senegalensis” was detected in only 16.8% of Rickettsia positive dog fleas. Sequence analysis of COI and COII revealed that almost all fleas collected from dogs were Ctenocephalides felis orientis. Other Rickettsia species were detected by NGS including Rickettsia heilongjiangensis from two Haemaphysalis hystricis ticks, and Rickettsia typhi in two rodent tissue samples. Conclusion: This study demonstrates the utility of NGS for high-throughput sequencing in the species characterization/identification of bacteria and ectoparasite for entomological surveillance of rickettsiae. A high percentage of C. f. orientis are positive for R. asembonensis. In addition, our findings indicate there is a risk of tick-borne Spotted Fever Group rickettsiosis, and flea-borne murine typhus transmission in Tak and Phangnga provinces of Thailand.

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Data analysis workflow for the detection of canine vector-borne pathogens using 16 S rRNA Next-Generation Sequencing

BMC Veterinary Research ◽

10.1186/s12917-021-02969-9 ◽

2021 ◽

Vol 17 (1) ◽

Author(s):

Elton J. R. Vasconcelos ◽

Chayan Roy ◽

Joseph A. Geiger ◽

Kristina M. Oney ◽

Melody Koo ◽

...

Keyword(s):

Veterinary Medicine ◽

Next Generation Sequencing ◽

Complete Analysis ◽

Molecular Diagnostic ◽

Rrna Gene ◽

Spotted Fever Group ◽

Next Generation ◽

Vector Borne ◽

16 S Rrna ◽

Generation Sequencing

Abstract Background Vector-borne diseases (VBDs) impact both human and veterinary medicine and pose special public health challenges. The main bacterial vector-borne pathogens (VBPs) of importance in veterinary medicine include Anaplasma spp., Bartonella spp., Ehrlichia spp., and Spotted Fever Group Rickettsia. Taxon-targeted PCR assays are the current gold standard for VBP diagnostics but limitations on the detection of genetically diverse organisms support a novel approach for broader detection of VBPs. We present a methodology for genetic characterization of VBPs using Next-Generation Sequencing (NGS) and computational approaches. A major advantage of NGS is the ability to detect multiple organisms present in the same clinical sample in an unsupervised (i.e. non-targeted) and semi-quantitative way. The Standard Operating Procedure (SOP) presented here combines industry-standard microbiome analysis tools with our ad-hoc bioinformatic scripts to form a complete analysis pipeline accessible to veterinary scientists and freely available for download and use at https://github.com/eltonjrv/microbiome.westernu/tree/SOP. Results We tested and validated our SOP by mimicking single, double, and triple infections in genomic canine DNA using serial dilutions of plasmids containing the entire 16 S rRNA gene sequence of (A) phagocytophilum, (B) v. berkhoffii, and E. canis. NGS with broad-range 16 S rRNA primers followed by our bioinformatics SOP was capable of detecting these pathogens in biological replicates of different dilutions. These results illustrate the ability of NGS to detect and genetically characterize multi-infections with different amounts of pathogens in a single sample. Conclusions Bloodborne microbiomics & metagenomics approaches may help expand the molecular diagnostic toolbox in veterinary and human medicine. In this paper, we present both in vitro and in silico detailed protocols that can be combined into a single workflow that may provide a significant improvement in VBP diagnostics and also facilitate future applications of microbiome research in veterinary medicine.

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Next-Generation Sequencing Identification and Characterization of MicroRNAs in Dwarfed Citrus Trees Infected With Citrus Dwarfing Viroid in High-Density Plantings

Frontiers in Microbiology ◽

10.3389/fmicb.2021.646273 ◽

2021 ◽

Vol 12 ◽

Author(s):

Tyler Dang ◽

Irene Lavagi-Craddock ◽

Sohrab Bodaghi ◽

Georgios Vidalakis

Keyword(s):

Next Generation Sequencing ◽

Target Genes ◽

Quantitative Polymerase Chain Reaction ◽

High Density ◽

Poncirus Trifoliata ◽

Next Generation ◽

Developmental Processes ◽

Wide Range ◽

Generation Sequencing ◽

Citrus Trees

Citrus dwarfing viroid (CDVd) induces stunting on sweet orange trees [Citrus sinensis (L.) Osbeck], propagated on trifoliate orange rootstock [Citrus trifoliata (L.), syn. Poncirus trifoliata (L.) Raf.]. MicroRNAs (miRNAs) are a class of non-coding small RNAs (sRNAs) that play important roles in the regulation of tree gene expression. To identify miRNAs in dwarfed citrus trees, grown in high-density plantings, and their response to CDVd infection, sRNA next-generation sequencing was performed on CDVd-infected and non-infected controls. A total of 1,290 and 628 miRNAs were identified in stem and root tissues, respectively, and among those, 60 were conserved in each of these two tissue types. Three conserved miRNAs (csi-miR479, csi-miR171b, and csi-miR156) were significantly downregulated (adjusted p-value < 0.05) in the stems of CDVd-infected trees compared to the non-infected controls. The three stem downregulated miRNAs are known to be involved in various physiological and developmental processes some of which may be related to the characteristic dwarfed phenotype displayed by CDVd-infected C. sinensis on C. trifoliata rootstock field trees. Only one miRNA (csi-miR535) was significantly downregulated in CDVd-infected roots and it was predicted to target genes controlling a wide range of cellular functions. Reverse transcription quantitative polymerase chain reaction analysis performed on selected miRNA targets validated the negative correlation between the expression levels of these targets and their corresponding miRNAs in CDVd-infected trees. Our results indicate that CDVd-responsive plant miRNAs play a role in regulating important citrus growth and developmental processes that may participate in the cellular changes leading to the observed citrus dwarf phenotype.

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355. A Novel Likelihood-Based Model to Estimate SARS-CoV-2 Viral Titer from Next-Generation Sequencing Data

Open Forum Infectious Diseases ◽

10.1093/ofid/ofab466.556 ◽

2021 ◽

Vol 8 (Supplement_1) ◽

pp. S281-S282

Author(s):

Heather L Wells ◽

Joseph Barrows ◽

Mara Couto-Rodriguez ◽

Xavier O Jirau Serrano ◽

Marilyne Debieu ◽

...

Keyword(s):

Next Generation Sequencing ◽

Board Member ◽

Clinical Specimen ◽

Next Generation Sequencing Data ◽

Viral Titer ◽

Next Generation ◽

Genome Coverage ◽

Ct Value ◽

Wide Range ◽

Generation Sequencing

Abstract Background The quantitative level of pathogens present in a host is a major driver of infectious disease (ID) state and outcome. However, the majority of ID diagnostics are qualitative. Next-generation sequencing (NGS) is an emerging ID diagnostics and research tool to provide insights, including tracking transmission, evolution, and identifying novel strains. Methods We built a novel likelihood-based computational method to leverage pathogen-specific genome-wide NGS data to detect SARS-CoV-2, profile genetic variants, and furthermore quantify levels of these pathogens. We used de-identified clinical specimens tested for SARS-CoV-2 using RT-PCR, SARS-CoV-2 NGS Assay (hybrid capture, Twist Bioscience), or ARTIC (amplicon-based) platform, and COVID-DX software. A training (n=87) and validation (n=22) set was selected to establish the strength of our quantification model. We fit non-uniform probabilistic error profiles to a deterministic sigmoidal equation that more realistically represents observed data and used likelihood maximized over several different read depths to improve accuracy over a wide range of values of viral load. Given the proportion of the genome covered at varying depths for a single sample as input data, our model estimated the Ct of that sample as the value that produces the maximum likelihood of generating the observed genome coverage data. Results The model fit on 87 SARS-CoV-2 NGS Assay training samples produced a good fit to the 22 validation samples, with a coefficient of correlation (r2) of ~0.8. The accuracy of the model was high (mean absolute % error of ~10%, meaning our model is able to predict the Ct value of each sample within a margin of ±10% on average). Because of the nature of the commonly used ARTIC protocol, we found that all quantitative signals in this data were lost during PCR amplification and the model is not applicable for quantification of samples captured this way. The ability to model quantification is a major advantage of the SARS-CoV-2 NGS assay protocol. The likelihood-based model to estimate SARS-CoV-2 viral titer Left Observed genome coverage (y-axis) plotted against Ct value (x-axis). The best-fitting logistic curve is demonstrated with a red line with shaded areas above and below representing the fitted error profile. RIGHT: Model-estimated Ct values (y-axis) compared to laboratory Ct values (x-axis) with grey bars representing estimated confidence intervals. The 1:1 diagonal is shown as a dotted line. Conclusion To our knowledge, this is the first model to incorporate sequence data mapped across the genome of a pathogen to quantify the level of that pathogen in a clinical specimen. This has implications in ID diagnostics, research, and metagenomics. Disclosures Heather L. Wells, MPH, Biotia, Inc. (Consultant) Joseph Barrows, MS, Biotia (Employee) Mara Couto-Rodriguez, MS, Biotia (Employee) Xavier O. Jirau Serrano, B.S., Biotia (Employee) Marilyne Debieu, PhD, Biotia (Employee) Karen Wessel, PhD, Labor Zotz/Klimas (Employee) Christopher Mason, PhD, Biotia (Board Member, Advisor or Review Panel member, Shareholder) Dorottya Nagy-Szakal, MD PhD, Biotia Inc (Employee, Shareholder) Niamh B. O’Hara, PhD, Biotia (Board Member, Employee, Shareholder)

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Multicenter validation study for the certification of a CFTR gene scanning method using next generation sequencing technology

Clinical Chemistry and Laboratory Medicine (CCLM) ◽

10.1515/cclm-2017-0553 ◽

2018 ◽

Vol 56 (7) ◽

pp. 1046-1053 ◽

Cited By ~ 9

Author(s):

Anne Bergougnoux ◽

Valeria D’Argenio ◽

Stefanie Sollfrank ◽

Fanny Verneau ◽

Antonella Telese ◽

...

Keyword(s):

Next Generation Sequencing ◽

Molecular Genetic Analysis ◽

Analytical Sensitivity ◽

Next Generation ◽

Sequencing Data ◽

Scanning Method ◽

Next Generation Sequencing Technology ◽

Wide Range ◽

Generation Sequencing

Abstract Background: Many European laboratories offer molecular genetic analysis of the CFTR gene using a wide range of methods to identify mutations causative of cystic fibrosis (CF) and CFTR-related disorders (CFTR-RDs). Next-generation sequencing (NGS) strategies are widely used in diagnostic practice, and CE marking is now required for most in vitro diagnostic (IVD) tests in Europe. The aim of this multicenter study, which involved three European laboratories specialized in CF molecular analysis, was to evaluate the performance of Multiplicom’s CFTR MASTR Dx kit to obtain CE-IVD certification. Methods: A total of 164 samples, previously analyzed with well-established “reference” methods for the molecular diagnosis of the CFTR gene, were selected and re-sequenced using the Illumina MiSeq benchtop NGS platform. Sequencing data were analyzed using two different bioinformatic pipelines. Annotated variants were then compared to the previously obtained reference data. Results and conclusions: The analytical sensitivity, specificity and accuracy rates of the Multiplicom CFTR MASTR assay exceeded 99%. Because different types of CFTR mutations can be detected in a single workflow, the CFTR MASTR assay simplifies the overall process and is consequently well suited for routine diagnostics.

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cDNA display coupled with next-generation sequencing for rapid activity-based screening: Comprehensive analysis of transglutaminase substrate preference

10.1101/2021.09.08.459404 ◽

2021 ◽

Author(s):

Jasmina Damnjanović ◽

Nana Odake ◽

Jicheng Fan ◽

Beixi Jia ◽

Takaaki Kojima ◽

...

Keyword(s):

Next Generation Sequencing ◽

Substrate Preference ◽

Stable Complex ◽

Next Generation ◽

Library Size ◽

Binary Model ◽

Wide Range ◽

Generation Sequencing ◽

Selection Of

AbstractcDNA display is an in vitro display technology based on a covalent linkage between a protein and its corresponding mRNA/cDNA, where a stable complex is formed suitable for a wide range of selection conditions. A great advantage of cDNA display is the ability to handle enormous library size (1012) in a microtube scale, in a matter of days. To harness its benefits, we aimed at developing a platform which combines the advantages of cDNA display with high-throughput and accuracy of next-generation sequencing (NGS) for the selection of preferred substrate peptides of transglutaminase 2 (TG2), a protein cross-linking enzyme. After the optimization of the platform by the repeated screening of binary model libraries consisting of the substrate and non-substrate peptides at different ratios, screening and selection of combinatorial peptide library randomized at positions -1, +1, +2, and +3 from the glutamine residue was carried out. Enriched cDNA complexes were analyzed by NGS and bioinformatics, revealing the comprehensive amino acid preference of the TG2 at targeted positions of the peptide backbone. This is the first report on the cDNA display/NGS screening system to yield comprehensive data on TG substrate preference. Although some issues remain to be solved, this platform can be applied to the selection of other TGs and easily adjusted for the selection of other peptide substrates and even larger biomolecules.

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Assessment of Antibody Library Diversity through Next Generation Sequencing and Technical Error Compensation.

10.1101/085498 ◽

2016 ◽

Author(s):

Marco Fantini ◽

Luca Pandolfini ◽

Simonetta Lisi ◽

Michele Chirichella ◽

Ivan Arisi ◽

...

Keyword(s):

Next Generation Sequencing ◽

Small Sample ◽

Sequencing Error ◽

Limited Information ◽

Next Generation ◽

Antibody Library ◽

Illumina Platform ◽

Wide Range ◽

Antibody Libraries ◽

Generation Sequencing

Antibody libraries are important resources to derive antibodies to be used for a wide range of applications, from structural and functional studies to intracellular protein interference studies to developing new diagnostics and therapeutics. Whatever the goal, the key parameter for an antibody library is its diversity, i.e. the number of distinct elements in the collection, which directly reflects the probability of finding in the library an antibody against a given antigen, of sufficiently high affinity. Quantitative evaluation of antibody library diversity and quality has been for a long time inadequately addressed, due to the high similarity and length of the sequences of the library. Diversity was usually inferred by the transformation efficiency and tested either by fingerprinting and/or sequencing of a few hundred random library elements. Inferring diversity from such a small sample is, however, very rudimental and gives limited information about the real complexity, because complexity does not scale linearly with sample size. Next-generation sequencing (NGS) has opened new ways to tackle the antibody library diversity quality assessment. However, much remains to be done to fully exploit the potential of NGS for the quantitative analysis of antibody repertoires and to overcome current limitations. To obtain a more reliable antibody library complexity estimate here we show a new, PCR-free, NGS approach to sequence antibody libraries on Illumina platform, coupled to a new bioinformatic analysis and software (Diversity Estimator of Antibody Library, DEAL) that allows to reliably estimate the diversity, taking in consideration the sequencing error.

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Next-generation sequencing in neuropathological diagnosis of infections of the nervous system

10.1101/039222 ◽

2016 ◽

Author(s):

Steven L. Salzberg ◽

Florian Breitwieser ◽

Anupama Kumar ◽

Haiping Hao ◽

Peter Burger ◽

...

Keyword(s):

Spinal Cord ◽

Nervous System ◽

Next Generation Sequencing ◽

Large Scale ◽

Infectious Agent ◽

Next Generation ◽

Dna And Rna ◽

Wide Range ◽

Next Generation Sequencing Ngs ◽

Generation Sequencing

Objective: To determine the feasibility of next-generation sequencing (NGS) microbiome approaches in the diagnosis of infectious disorders in brain or spinal cord biopsies in patients with suspected central nervous system (CNS) infections. Methods: In a prospective-pilot study, we applied NGS in combination with a new computational analysis pipeline to detect the presence of pathogenic microbes in brain or spinal cord biopsies from ten patients with neurological problems indicating possible infection but for whom conventional clinical and microbiology studies yielded negative or inconclusive results. Results: Direct DNA and RNA sequencing of brain tissue biopsies generated 8.3 million to 29.1 million sequence reads per sample, which successfully identified with high confidence the infectious agent in three patients, identified possible pathogens in two more, and helped to understand neuropathological processes in three others, demonstrating the power of large-scale unbiased sequencing as a novel diagnostic tool. Validation techniques confirmed the pathogens identified by NGS in each of the three positive cases. Clinical outcomes were consistent with the findings yielded by NGS on the presence or absence of an infectious pathogenic process in eight of ten cases, and were non-contributory in the remaining two. Conclusions: NGS-guided metagenomic studies of brain, spinal cord or meningeal biopsies offer the possibility for dramatic improvements in our ability to detect (or rule out) a wide range of CNS pathogens, with potential benefits in speed, sensitivity, and cost. NGS-based microbiome approaches present a major new opportunity to investigate the potential role of infectious pathogens in the pathogenesis of neuroinflammatory disorders.

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Study of infection by Rickettsiae of the spotted fever group in humans and ticks in an urban park located in the City of Londrina, State of Paraná, Brazil

Revista da Sociedade Brasileira de Medicina Tropical ◽

10.1590/s0037-86822011005000037 ◽

2011 ◽

Vol 44 (3) ◽

pp. 313-317 ◽

Cited By ~ 2

Author(s):

Roberta Santos Toledo ◽

Katia Tamekuni ◽

Mauro de Freitas Silva Filho ◽

Valeska Bender Haydu ◽

Richard Campos Pacheco ◽

...

Keyword(s):

Citrate Synthase ◽

Spotted Fever ◽

Immunofluorescence Assay ◽

Etiologic Agent ◽

Spotted Fever Group ◽

Rickettsia Species ◽

Rickettsia Rickettsii ◽

Serological Studies ◽

Human Sera ◽

Brazilian Spotted Fever

INTRODUCTION: Spotted fevers are emerging zoonoses caused by Rickettsia species in the spotted fever group (SFG). Rickettsia rickettsii is the main etiologic agent of Brazilian spotted fever (BSF) and it is transmitted by Amblyomma spp. ticks. METHODS: The study aimed to investigate SFG rickettsiae in the Arthur Thomas Municipal Park in Londrina, PR, by collecting free-living ticks and ticks from capybaras and blood samples from personnel working in these areas. Samples from A. dubitatum and A. cajennense were submitted for PCR in pools to analyze the Rickettsia spp. gltA (citrate synthase gene). RESULTS: All the pools analyzed were negative. Human sera were tested by indirect immunofluorescence assay with R. rickettsii and R. parkeri as antigens. Among the 34 sera analyzed, seven (20.6%) were reactive for R. rickettsii: four of these had endpoint titers equal to 64, 2 titers were 128 and 1 titer was 256. None of the samples were reactive for R. parkeri. An epidemiological questionnaire was applied to the park staff, but no statistically significant associations were identified. CONCLUSIONS: The serological studies suggest the presence of Rickettsiae related to SFG that could be infecting the human population studied; however, analysis of the ticks collected was unable to determine which species may be involved in transmission to humans.

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Leafhopper Viral Pathogens: Ultrastructure of salivary gland infection of Taastrup-like virus inPsammotettix alienusDahlbom; and a novel Rhabdovirus inHomalodisca vitripennis(Germar) Hemiptera: Cicadellidae

10.1101/465468 ◽

2018 ◽

Author(s):

Thorben Lundsgaard ◽

Wayne B. Hunter ◽

Scott Adkins

Keyword(s):

Next Generation Sequencing ◽

Plant Pathogens ◽

Insect Pests ◽

Economic Losses ◽

Type I ◽

Next Generation ◽

Viral Pathogen ◽

Thin Sections ◽

Wide Range ◽

Generation Sequencing

AbstractViruses that are pathogenic to insect pests can be exploited as biological control agents. Viruses that are pathogenic to beneficial insects and other arthropods, as in honey bees, silk worms, and shrimp, cause millions of dollars of losses to those industries. Current advances in next generation sequencing technologies along with molecular and cellular biology have produced a wealth of information about insect viruses and their potential applications. Leafhoppers cause economic losses as vectors of plant pathogens which significantly reduce the worlds’ food crops. Each year more viruses are discovered primarily through the use of next generation sequencing of the leafhopper hosts. The diversity of viruses from leafhoppers demonstrates a wide range of taxonomic members that includes genomes of DNA or RNA from families like: Reoviridae, Iridoviridae, Dicistroviridae, Iflaviridae, and others yet to be classified. Discussed is a recent viral pathogen isolated from the leafhopperPsammotettix alienus, name Taastrup Virus. Taastrup virus (TV) is a novel virus with a RNA genome, a Filovirus-like morphology, being tentatively placed within theMononegavirales. AdultPsammotettix alienusinfected with TV, showed the highest concentration of virions in salivary glands, consisting of a principal gland (type I-VI-cells) and an accessory gland. Examination of thin sections revealed enveloped particles, about 1300 nm long and 62 nm in diameter, located singly or in paracrystalline arrays in canaliculi of type III- and IV-cells. In gland cells with TV particles in canaliculi, granular masses up to 15 μm in diameter were present in the cytoplasm. These masses are believed to be viroplasms, the sites for viral replication. TV particles were observed at the connection between a canaliculus and the salivary duct system. A TV-like virus with strongly similar morphology was discovered in the ornamental plant,Liriope, near Fort Pierce, Florida, USA. When the virus was inoculated to a leafhopper cell culture, HvWH, made from the glassy-winged sharpshooter,Homalodisca vitripennis(Germar), the cells rapidly degraded with 100% mortality in 48 hours. These two instances are the only reported cases of this newly discovered viral pathogen of leafhoppers.

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

BMC Bioinformatics ◽

10.1186/s12859-021-04545-2 ◽

2022 ◽

Vol 23 (1) ◽

Author(s):

Ludwig Mann ◽

Kathrin M. Seibt ◽

Beatrice Weber ◽

Tony Heitkam

Keyword(s):

Next Generation Sequencing ◽

Transposable Elements ◽

Data Availability ◽

Next Generation Sequencing Data ◽

Next Generation ◽

Sequencing Data ◽

Bioinformatics Pipeline ◽

Circular Dna ◽

Wide Range ◽

Generation Sequencing

Abstract 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.

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