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

2021 ◽  
Author(s):  
Ludwig Mann ◽  
Kathrin M. Seibt ◽  
Beatrice Weber ◽  
Tony Heitkam
Keyword(s):  
Next Generation Sequencing ◽  
Transposable Elements ◽  
Control Sample ◽  
Data Availability ◽  
Next Generation Sequencing Data ◽  
Model Organisms ◽  
Next Generation ◽  
Circular Dna ◽  
Wide Range ◽  
Generation Sequencing

Motivation: 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 H. sapiens and A. thaliana, and generated circSeq reads from the non-model crop B. 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. 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. Availability and implementation: The ECCsplorer pipeline is available on GitHub at https://github.de/crimBubble/ECCsplorer under the GNU license.

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

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.

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

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)

scholarly journals Microsatellite loci discovery from next-generation sequencing data and loci characterization in the epizoic barnacleChelonibia testudinaria(Linnaeus, 1758)

PeerJ ◽  
2016 ◽  
Vol 4 ◽  
pp. e2019 ◽  
Author(s):  
Christine Ewers-Saucedo ◽  
John D. Zardus ◽  
John P. Wares
Keyword(s):  
Next Generation Sequencing ◽  
Microsatellite Markers ◽  
Microsatellite Loci ◽  
Next Generation Sequencing Data ◽  
Model Organisms ◽  
Next Generation ◽  
Sequencing Data ◽  
Genetic Studies ◽  
Evolutionary Features ◽  
Generation Sequencing

Microsatellite markers remain an important tool for ecological and evolutionary research, but are unavailable for many non-model organisms. One such organism with rare ecological and evolutionary features is the epizoic barnacleChelonibia testudinaria(Linnaeus, 1758).Chelonibia testudinariaappears to be a host generalist, and has an unusual sexual system, androdioecy. Genetic studies on host specificity and mating behavior are impeded by the lack of fine-scale, highly variable markers, such as microsatellite markers. In the present study, we discovered thousands of new microsatellite loci from next-generation sequencing data, and characterized 12 loci thoroughly. We conclude that 11 of these loci will be useful markers in future ecological and evolutionary studies onC. testudinaria.

scholarly journals T-lex2: genotyping, frequency estimation and re-annotation of transposable elements using single or pooled next-generation sequencing data

2014 ◽  
Author(s):  
Anna-Sophie Fiston-Lavier ◽  
Maite G. Barrón ◽  
Dmitri A. Petrov ◽  
Josefa González
Keyword(s):  
Next Generation Sequencing ◽  
Transposable Elements ◽  
Frequency Estimation ◽  
Next Generation Sequencing Data ◽  
Next Generation ◽  
Sequencing Data ◽  
Flexible Tool ◽  
Depth Analysis ◽  
Individual Strain ◽  
Generation Sequencing

Transposable elements (TEs) constitute the most active, diverse and ancient component in a broad range of genomes. Complete understanding of genome function and evolution cannot be achieved without a thorough understanding of TE impact and biology. However, in-depth analysis of TEs still represents a challenge due to the repetitive nature of these genomic entities. In this work, we present a broadly applicable and flexible tool: T-lex2. T-lex2 is the only available software that allows routine, automatic, and accurate genotyping of individual TE insertions and estimation of their population frequencies both using individual strain and pooled next-generation sequencing (NGS) data. Furthermore, T-lex2 also assesses the quality of the calls allowing the identification of miss-annotated TEs and providing the necessary information to re-annotate them. The flexible and customizable design of T-lex2 allows running it in any genome and for any type of TE insertion. Here, we tested the fidelity of T-lex2 using the fly and human genomes. Overall, T-lex2 represents a significant improvement in our ability to analyze the contribution of TEs to genome function and evolution as well as learning about the biology of TEs. T-lex2 is freely available online at http://sourceforge.net/projects/tlex/.

scholarly journals Microsatellite loci discovery from next-generation sequencing data and marker characterization in the epizoic barnacle Chelonibia testudinaria (Linnaeus, 1798)

2016 ◽  
Author(s):  
Christine Ewers-Saucedo ◽  
John D Zardus ◽  
John P Wares
Keyword(s):  
Next Generation Sequencing ◽  
Microsatellite Loci ◽  
Next Generation Sequencing Data ◽  
Model Organisms ◽  
Next Generation ◽  
Sequencing Data ◽  
Genetic Studies ◽  
Evolutionary Features ◽  
Evolutionary Studies ◽  
Generation Sequencing

Microsatellite markers remain an important tool for ecological and evolutionary research, but are unavailable for many non-model organisms. One such organism with rare ecological and evolutionary features is the epizoic barnacle Chelonibia testudinaria (Linnaeus, 1758). Chelonibia testudinaria appears to be a host generalist, and has a unusual sexual system, androdioecy. Genetic studies on host specificity and mating behavior are impeded by the lack of fine-scale, highly variable markers. In the present study, we discovered thousands of new microsatellite loci from next-generation sequencing data, and characterized 12 loci thoroughly. We conclude that 11 of these loci will be useful markers in future ecological and evolutionary studies on C. testudinaria.

scholarly journals easyfm: An easy software suite for file manipulation of Next Generation Sequencing data on desktops

2021 ◽  
Author(s):  
Hyungtaek Jung ◽  
Brendan Jeon ◽  
Daniel Ortiz-Barrientos
Keyword(s):  
Next Generation Sequencing ◽  
Life Sciences ◽  
Next Generation Sequencing Data ◽  
Command Line ◽  
Next Generation ◽  
Web Based ◽  
File Formats ◽  
Wide Range ◽  
Ngs Data ◽  
Generation Sequencing

Storing and manipulating Next Generation Sequencing (NGS) file formats for understanding biological phenomena is an essential but difficult task in the life sciences. Yet, most methods for analysing NGS data require complex command-line tools in high-performance computing (HPC) or web-based servers and have not yet been implemented in comprehensive, easy-to-use software. Here we present easyfm (easy file manipulation), a free standalone Graphical User Interface (GUI) software with Python support that can be used to facilitate the rapid discovery of target sequences (or user’s interest) in NGS datasets for novice users (more accessible to biologists). It enables them to perform end-to-end reproducible data analyses using a desktop application (Windows, Mac and Linux). Unlike existing tools, the GUI-based easyfm is not dependent on any HPC system and can be operated without an internet connection. For user-friendliness and convenience, easyfm was developed with four work modules and a secondary GUI window, covering different aspects of NGS data analysis, including post-processing, filtering, format conversion, generating results, real-time log, and help. In combination with the executable tools (BLAST+ and BLAT) and Python, easyfm allows the user to set analysis parameters, select/extract regions of interest, examine the input and output results, and convert to a wide range of file formats. To help augment the functionality of existing web-based and command-line tools, easyfm, a self-contained program, comes with extensive documentation (https://github.com/TaekAndBrendan/easyfm). This specific benefit allows easyfm to seamlessly integrate visual and interactive representations of NGS files, supporting a wider scope of bioinformatics applications in the life sciences.

scholarly journals Microsatellite marker discovery from next-generation sequencing data and subsequent marker characterization in the epizoic barnacle Chelonibia testudinaria (Linnaeus, 1798)

2016 ◽  
Author(s):  
Christine Ewers-Saucedo ◽  
John D Zardus ◽  
John P Wares
Keyword(s):  
Next Generation Sequencing ◽  
Next Generation Sequencing Data ◽  
Model Organisms ◽  
Next Generation ◽  
Sequencing Data ◽  
Genetic Studies ◽  
Evolutionary Features ◽  
Evolutionary Studies ◽  
Marker Discovery ◽  
Generation Sequencing

Microsatellite markers remain an important tool for ecological and evolutionary research, but are unavailable for many non-model organisms. One such organism with rare ecological and evolutionary features is the epizoic barnacle Chelonibia testudinaria (Linnaeus, 1758). Chelonibia testudinaria appears to be a host generalist, and has a unusual sexual system, androdioecy. Genetic studies on host specificity and mating behavior are impeded by the lack of fine-scale, highly variable markers. In the present study, we discovered thousands of new microsatellite loci from next-generation sequencing data, and characterized 12 loci thoroughly. We conclude that 11 of these loci will be useful markers in future ecological and evolutionary studies on C. testudinaria.

scholarly journals Microsatellite loci discovery from next-generation sequencing data and marker characterization in the epizoic barnacle Chelonibia testudinaria (Linnaeus, 1798)

2016 ◽  
Author(s):  
Christine Ewers-Saucedo ◽  
John D Zardus ◽  
John P Wares
Keyword(s):  
Next Generation Sequencing ◽  
Microsatellite Loci ◽  
Next Generation Sequencing Data ◽  
Model Organisms ◽  
Next Generation ◽  
Sequencing Data ◽  
Genetic Studies ◽  
Evolutionary Features ◽  
Evolutionary Studies ◽  
Generation Sequencing

Microsatellite markers remain an important tool for ecological and evolutionary research, but are unavailable for many non-model organisms. One such organism with rare ecological and evolutionary features is the epizoic barnacle Chelonibia testudinaria (Linnaeus, 1758). Chelonibia testudinaria appears to be a host generalist, and has a unusual sexual system, androdioecy. Genetic studies on host specificity and mating behavior are impeded by the lack of fine-scale, highly variable markers. In the present study, we discovered thousands of new microsatellite loci from next-generation sequencing data, and characterized 12 loci thoroughly. We conclude that 11 of these loci will be useful markers in future ecological and evolutionary studies on C. testudinaria.

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