Circular Sequence Research Articles

Experimental Tests of the Virtual Circular Genome Model for Nonenzymatic RNA Replication.

The virtual circular genome (VCG) model was proposed as a means of going beyond template copying to indefinite cycles of nonenzymatic RNA replication during the origin of life. In the VCG model, the protocellular genome is a collection of short oligonucleotides that map to both strands of a virtual circular sequence. Replication is driven by templated nonenzymatic primer extensions on a subset of kinetically trapped partially base-paired configurations, followed by the shuffling of these configurations to enable continued oligonucleotide elongation. Here, we describe initial experimental studies of the feasibility of the VCG model for replication. We designed a small 12-nucleotide model VCG and synthesized all 247 oligonucleotides of lengths 2 to 12 corresponding to this genome. We experimentally monitored the fate of individual labeled primers in the pool of VCG oligonucleotides following the addition of activated nucleotides and investigated the effect of factors such as oligonucleotide length, concentration, composition, and temperature on the extent of primer extension. We observe a surprisingly prolonged equilibration process in the VCG system that enables a considerable extent of reaction. We find that environmental fluctuations would be essential for continuous templated extension of the entire VCG system since the shortest oligonucleotides can only bind to templates at low temperatures, while the longest oligonucleotides require high-temperature spikes to escape from inactive configurations. Finally, we demonstrate that primer extension is significantly enhanced when the mix of VCG oligonucleotides is preactivated. We discuss the necessity of ongoing in situ activation chemistry for continuous and accurate VCG replication.

Assessing the impacts of various factors on circular RNA reliability.

Circular RNAs (circRNAs) are non-polyadenylated RNAs with a continuous loop structure characterized by a non-colinear back-splice junction (BSJ). Although millions of circRNA candidates have been identified, it remains a major challenge for determining circRNA reliability because of various types of false positives. Here, we systematically assess the impacts of numerous factors related to circRNA identification, conservation, biogenesis, and function on circRNA reliability by comparisons of circRNA expression from mock and the corresponding colinear/polyadenylated RNA-depleted datasets based on three different RNA treatment approaches. Eight important indicators of circRNA reliability are determined. The relative contribution to variability explained analyses reveal that the relative importance of these factors in affecting circRNA reliability in descending order is the conservation level of circRNA, full-length circular sequences, supporting BSJ read count, both BSJ donor and acceptor splice sites at the same colinear transcript isoforms, both BSJ donor and acceptor splice sites at the annotated exon boundaries, BSJs detected by multiple tools, supporting functional features, and both BSJ donor and acceptor splice sites undergoing alternative splicing. This study thus provides a useful guideline and an important resource for selecting high-confidence circRNAs for further investigations.

Complete Genome Sequences of Three Salmonella Strains Obtained from a Poultry Production Farm in Thailand.

Here, we report three complete circular genome sequences of Salmonella enterica SalSpp07, SalSpp08, and SalSpp09, which were isolated from chicken meat and skin during quality control on the production line. The genomes were closed using a hybrid assembly method with short and long sequencing reads.

Complete mitochondrial genome of the Spanish toothcarp, Aphanius iberus (Valenciennes, 1846) (Actinopterygii, Aphaniidae) and its phylogenetic position within the Cyprinodontiformes order

BackgroundThe Spanish toothcarp (Aphanius iberus Valenciennes, 1846) is a small fish endemic to the eastern coastline of the Iberian Peninsula and is currently listed as “Endangered” (category IUCN: EN). It mainly inhabits brackish waters which can exhibit large fluctuations in temperature and salinity throughout the year. The genetics of A. iberus are not well-known since most studies have only evaluated the genetic structure of the species under a conservation framework in order to identify its potential conservation units. Different phylogenetic relationships of Aphanius have been published based on some particular genes. In the present study, the entire mitochondrial genome of A. iberus was obtained for the first time in the context of an A. iberus reference genome and a hypothesis regarding its phylogenetic position was considered.Methods and resultsThe mitogenome (a circular doble-stranded DNA sequence of 16,708 bp) was reconstructed and aligned against 83 Cyprinodontiformes and two outgroup taxa to identify the phylogenetic position of A. iberus. PartitionFinder was first used to test for the best evolutionary model and the phylogenetic analyses were performed using two methods: Maximun-Likelihood Approximation (IQ-Tree) and Bayesian inference (MrBayes). Our results show that A. iberus forms a sister group with Orestias ascotanensis, a cyprinodontiform species native to South America.ConclusionsThe results were congruent with the traditional morphometric reconstructed trees and with a geological vicariant hypothesis involving Cyprinodontiformes where Aphaniidae is shown as a monophyletic family separated from the family Cyprinodontidae. The information gathered from this study is not only valuable for improving our understanding of the evolutionary history of A. iberus, but for future genomic studies involving the species.

Mapping DNA Conformations Using Single-Molecule Conductance Measurements.

DNA is an attractive material for a range of applications in nanoscience and nanotechnology, and it has recently been demonstrated that the electronic properties of DNA are uniquely sensitive to its sequence and structure, opening new opportunities for the development of electronic DNA biosensors. In this report, we examine the origin of multiple conductance peaks that can occur during single-molecule break-junction (SMBJ)-based conductance measurements on DNA. We demonstrate that these peaks originate from the presence of multiple DNA conformations within the solutions, in particular, double-stranded B-form DNA (dsDNA) and G-quadruplex structures. Using a combination of circular dichroism (CD) spectroscopy, computational approaches, sequence and environmental controls, and single-molecule conductance measurements, we disentangle the conductance information and demonstrate that specific conductance values come from specific conformations of the DNA and that the occurrence of these peaks can be controlled by controlling the local environment. In addition, we demonstrate that conductance measurements are uniquely sensitive to identifying these conformations in solutions and that multiple configurations can be detected in solutions over an extremely large concentration range, opening new possibilities for examining low-probability DNA conformations in solutions.

Circular RNAs in and out of Cells: Therapeutic Usages of Circular RNAs.

RNAs are versatile molecules that are primarily involved in gene regulation and can thus be widely used to advance the fields of therapeutics and diagnostics. In particular, circular RNAs which are highly stable, have emerged as strong candidates for use on next-generation therapeutic platforms. Endogenous circular RNAs control gene regulatory networks by interacting with other biomolecules or through translation into polypeptides. Circular RNAs exhibit cell-type specific expression patterns, which can be altered in tissues and body fluids depending on pathophysiological conditions. Circular RNAs that are aberrantly expressed in diseases can function as biomarkers or therapeutic targets. Moreover, exogenous circular RNAs synthesized in vitro can be introduced into cells as therapeutic molecules to modulate gene expression networks in vivo. Depending on the purpose, synthetic circular RNA sequences can either be identical to endogenous circular RNA sequences or artificially designed. In this review, we introduce the life cycle and known functions of intracellular circular RNAs. The current stage of endogenous circular RNAs as biomarkers and therapeutic targets is also described. Finally, approaches and considerations that are important for applying the available knowledge on endogenous circular RNAs to design exogenous circular RNAs for therapeutic purposes are presented.

Optimal non‐circular designs for total effects under interference models

In block designs, the responses of plots are potentially influenced by the treatments of neighbouring plots and the surrounding environment. Many researchers use two guarding plots next to the edge plots, for which we apply certain treatments to control these environmental effects. Thus, a design is presented as a collection of treatment sequences. For the estimation of total effects, existing results consider circular designs, whose constraints are unnecessary in common applications. In this paper, we construct optimal or highly efficient non‐circular designs under interference models. It is observed that the optimal non‐circular designs for the total effects outperform the optimal circular designs in many instances. In fact, a design containing a circular sequence cannot be optimal for .

Inverse PCR for Site-Directed Mutagenesis.

Inverse PCR is a powerful tool for the rapid introduction of desired mutations at desired positions in a circular double-stranded DNA sequence. In this technique, custom-designed mutant primers oriented in the inverse direction are used to amplify the entire circular template with incorporation of the required mutation(s). By careful primer design, it can be used to perform such diverse modifications as the introduction of point or multiple mutations, the insertion of new sequences, and even sequence deletions. Three primer formats are commonly used, nonoverlapping, partially overlapping, and fully overlapping primers, and here we describe the use of nonoverlapping primers for introduction of a point mutation. Use of such a primer setup in the PCR, with one of the primers containing the desired mismatch mutation, results in the amplification of a linear, double-stranded, mutated product. Methylated template DNA is removed from the non-methylated PCR product by DpnI digestion, and the PCR product is then phosphorylated by polynucleotide kinase treatment before being recircularized by ligation and transformed to E. coli. This relatively simple site-directed mutagenesis procedure is of major importance in biology and biotechnology where it is commonly employed for the study and engineering of DNA, RNA, and proteins.

Роль самопроектирования в формировании готовности будущего специалиста к профессиональному росту

The exclusive role of self-design enables life and professional planning in adolescence. The basis of the qualitative preparation of a future specialist for professional activity is a set of special tools and methods integrated into the system of formation of a person’s readiness to carry out professional development processes. Self-designing is a kind of self-education and self-realization program that has goals and contains a description of the means to achieve them. This allows to actively and consciously build a professional perspective career. The process of self-designing in relation to the construction of professional prospects is a circular sequence of interrelated stages. Among named components of the readiness for successful professional activity, there are theoretical readiness, psychological readiness, practical readiness. The readiness of the individual for self-improvement is fundamental for a professional career and further professional growth. We analyze results of self-design influence on the formation of students’ readiness for professional growth. The analysis present that the purposeful, systematic inclusion of students in self-design activities provides the practical experience and skills required to build professional prospects. It is concluded that self-design enables the individual to systematically carry out his life activity and is one of the components of the future specialist's readiness for professional growth.

An improved method of job shop scheduling using machine learning and mathematical optimization

This research is concerned with finding the optimal solution of a job shop scheduling problem (JSSP) in as short time as possible. A JSSP can be formulated as a 0-1 mixed integer programming (0-1 MIP), and it is expected that the optimal schedule can be obtained in a shorter time by predicting a good solution based on data of scheduling performed previously and using the solution as the initial solution for the optimization algorithm. In this concept, it is an important point to predict a solution which satisfy constraints of the 0-1 MIP. This paper provides an improved method based on this concept where the prediction is carried out so that the constraints are always satisfied. Numerical experiments showed that solution time of the proposed method is shorter than that of the previous method, which does not assure the constraints are satisfied, and learning time reduces about half. In addition, it turned out that there is a reason of the shorter solution time other than that the predicted solution always satisfies the constraints. One possible reason is that the number of circular sequences tends to be smaller than the previous method. Further analysis and additional evaluations from this point of view will be performed in a future work.

Identification of a conserved maxicircle and unique minicircles as part of the mitochondrial genome of Leishmania martiniquensis strain PCM3 in Thailand

BackgroundThe mitochondrial DNA of trypanosomatids, including Leishmania, is known as kinetoplast DNAs (kDNAs). The kDNAs form networks of hundreds of DNA circles that are evidently interlocked and require complex RNA editing. Previous studies showed that kDNA played a role in drug resistance, adaptation, and survival of Leishmania. Leishmania martiniquensis is one of the most frequently observed species in Thailand, and its kDNAs have not been illustrated.MethodsThis study aimed to extract the kDNA sequences from Illumina short-read and PacBio long-read whole-genome sequence data of L. martiniquensis strain PCM3 priorly isolated from the southern province of Thailand. A circular maxicircle DNA was reconstructed by de novo assembly using the SPAdes program, while the minicircle sequences were retrieved and assembled by the rKOMIC tool. The kDNA contigs were confirmed by blasting to the NCBI database, followed by comparative genomic and phylogenetic analysis.ResultsWe successfully constructed the complete circular sequence of the maxicircle (19,008 bp) and 214 classes of the minicircles from L. martiniquensis strain PCM3. The genome comparison and annotation showed that the maxicircle structure of L. martiniquensis strain PCM3 was similar to those of L. enriettii strain LEM3045 (84.29%), L. arabica strain LEM1108 (82.79%), and L. tarentolae (79.2%). Phylogenetic analysis also showed unique evolution of the minicircles of L. martiniquensis strain PCM3 from other examined Leishmania species.ConclusionsThis was the first report of the complete maxicircle and 214 minicircles of L. martiniquensis strain PCM3 using integrated whole-genome sequencing data. The information will be helpful for further improvement of diagnosis methods and monitoring genetic diversity changes of this parasite.Graphical abstract

Polymeric structure of the Cannabis sativa L. mitochondrial genome identified with an assembly graph model

Cannabis sativa L. belongs to the family Cannabaceae in Rosales. It has been widely used as medicines, building materials, and textiles. Elucidating its genome is critical for molecular breeding and synthetic biology study. Many studies have shown that the mitochondrial genomes (mitogenomes) and even chloroplast genomes (plastomes) had complex polymeric structures. Using the Nanopore sequencing platform, we sequenced, assembled, and analyzed its mitogenome and plastome. The resulting unitig graph suggested that the mitogenome had a complex polymeric structure. However, a gap-free, circular sequence was further assembled from the unitig graph. In contrast, a circular sequence representing the plastome was obtained. The mitogenome major conformation was 415,837 bp long, and the plastome was 153,927 bp long. To test if the repeat sequences promote recombination, which corresponds to the branch points in the structure, we tested the sequences around repeats by long-read mapping. Among 208 pairs of predicted repeats, the mapping results supported the presence of cross-over around 25 pairs of repeats. Subsequent PCR amplification confirmed the presence of cross-over around 15 of the 25 repeats. By comparing the mitogenome and plastome sequences, we identified 19 mitochondria plastid DNAs, including seven complete genes (trnW-CCA, trnP-UGG, psbJ, trnN-GUU, trnD-GUC, trnH-GUG, trnM-CAU) and nine gene fragments. Furthermore, the selective pressure analysis results showed that five genes (atp1, ccmB, ccmC, cox1, nad7) had 19 positively selected sites. Lastly, we predicted 28 RNA editing sites. A total of 8 RNA editing sites located in the coding regions were successfully validated by PCR amplification and Sanger sequencing, of which four were synonymous, and four were nonsynonymous. In particular, the RNA editing events appeared to be tissue-specific in C. sativa mitogenome. In summary, we have confirmed the major confirmation of C. sativa mitogenome and characterized its structural features in detail. These results provide critical information for future variety breeding and resource development for C. sativa.

REVIEW ARTICLE: HCV IMPACTON THE PATTERN OF CIRC-RNAEXPRESSION IN HCV RELATED HCC

Circular RNAs (circRNAs) are a class of non-coding RNAs with a circular, covalent structure, which are highly conserved, stable unique molecules found in eukaryotic cells. The most notable character of circRNAs is their high stability in biological systems which is a key factor in their potential for use in a variety of RNA-focused medical applications. Despite the fact that circRNAs have a wide range of biological roles and regulatory functions, their circular structure and sequence overlap with their linear mRNA counterparts make it difficult to research them in depth. Furthermore, little is known about their function in viral infections and in immune responses.Since circRNAs have been found to be involved in a number of viral infections (including hepatitis B virus infection and human papilloma virus infection), their significance in viral infections is being more recognized in the last years.In this review, we aimed to provide a broad basis and overview on the biogenesis, significance and regulatory roles of circRNAs in the context of viral hepatitis C virus (HCV) infection, and HCV related hepatocellular carcinoma. Owing to the fact that the chronic HCV infection is the leading cause of HCC in the western countries especially Egypt. HCV can act directly on cell signaling pathways to promote HCC by inhibiting tumor suppressor genes or by causing activation of signaling pathways that up-regulate growth and division. Because viruses frequently manipulate cellular pathways to control viral gene expression, cellular and viral circRNA landscapes in virus-infected cells are altered. Studying the contribution of circRNAs in these pathways may give valuable information in the pathogenesis of HCC caused by HCV or in finding more reliable therapeutic approaches.

CircSeqAlignTk: An R package for end-to-end analysis of RNA-seq data for circular genomes.

RNA sequencing (RNA-seq) technology has become one of the standard tools for studying biological mechanisms at the transcriptome level. Advances in RNA-seq technology have led to the development of numerous publicly available tools for RNA-seq data analysis. Most of these tools target linear genome sequences despite the necessity of studying organisms with circular genome sequences. For example, studying the infection mechanisms of viroids which comprise 246-401 nucleotides circular RNAs and target plants may prevent tremendous economic and agricultural damage. Unfortunately, using the available tools to construct workflows for the analysis of circular genome sequences is difficult, especially for non-bioinformaticians. To overcome this limitation, we present CircSeqAlignTk, an easy-to-use and richly documented R package. CircSeqAlignTk offers both command line and graphical user interfaces for end-to-end RNA-seq data analysis, spanning alignment to the visualisation of circular genome sequences, via a series of functions. Moreover, it includes a feature to generate synthetic sequencing data that mirrors real RNA-seq data from biological experiments. CircSeqAlignTk not only provides an easy-to-use analysis interface for novice users but also allows developers to evaluate the performance of alignment tools and new workflows.

The complete organellar genomes of the entheogenic plant Psychotria viridis (Rubiaceae), a main component of the ayahuasca brew.

Psychotria viridis (Rubioideae: Rubiaceae), popularly known as chacrona, is commonly found as a shrub in the Amazon region and is well-known to produce psychoactive compounds, such as the N,N-dimethyltryptamine (DMT). Together with the liana Banisteropsis caapi, P. viridis is one of the main components of the Amerindian traditional, entheogenic beverage known as ayahuasca. In this work, we assembled and annotated the organellar genomes (ptDNA and mtDNA), presenting the first genomics resources for this species. The P. viridis ptDNA exhibits 154,106 bp, encoding all known ptDNA gene repertoire found in angiosperms. The Psychotria genus is a complex paraphyletic group, and according to phylogenomic analyses, P. viridis is nested in the Psychotrieae clade. Comparative ptDNA analyses indicate that most Rubiaceae plastomes present conserved ptDNA structures, often showing slight differences at the junction sites of the major four regions (LSC-IR-SSC). For the mitochondrion, assembly graph-based analysis supports a complex mtDNA organization, presenting at least two alternative and circular mitogenomes structures exhibiting two main repeats spanning 24 kb and 749 bp that may symmetrically isomerize the mitogenome into variable arrangements and isoforms. The circular mtDNA sequences (615,370 and 570,344 bp) encode almost all plant mitochondrial genes (except for the ccmC, rps7, rps10, rps14, rps19, rpl2 and rpl16 that appears as pseudogenes, and the absent genes sdh3, rps2, rsp4, rsp8, rps11, rpl6, and rpl10), showing slight variations related to exclusive regions, ptDNA integration, and relics of previous events of LTR-RT integration. The detection of two mitogenomes haplotypes is evidence of heteroplasmy as observed by the complex organization of the mitochondrial genome using graph-based analysis. Taken together, these results elicit the primary insights into the genome biology and evolutionary history of Psychotria viridis and may be used to aid strategies for conservation of this sacred, entheogenic species.

Full-length transcriptome analysis of the bloom-forming dinoflagellate Akashiwo sanguinea by single-molecule real-time sequencing.

The dinoflagellate Akashiwo sanguinea is a harmful algal species and commonly observed in estuarine and coastal waters around the world. Harmful algal blooms (HABs) caused by this species lead to serious environmental impacts in the coastal waters of China since 1998 followed by huge economic losses. However, the full-length transcriptome information of A. sanguinea is still not fully explored, which hampers basic genetic and functional studies. Herein, single-molecule real-time (SMRT) sequencing technology was performed to characterize the full-length transcript in A. sanguinea. Totally, 83.03 Gb SMRT sequencing clean reads were generated, 983,960 circular consensus sequences (CCS) with average lengths of 3,061 bp were obtained, and 81.71% (804,016) of CCS were full-length non-chimeric reads (FLNC). Furthermore, 26,461 contigs were obtained after being corrected with Illumina library sequencing, with 20,037 (75.72%) successfully annotated in the five public databases. A total of 13,441 long non-coding RNA (lncRNA) transcripts, 3,137 alternative splicing (AS) events, 514 putative transcription factors (TFs) members from 23 TF families, and 4,397 simple sequence repeats (SSRs) were predicted, respectively. Our findings provided a sizable insights into gene sequence characteristics of A. sanguinea, which can be used as a reference sequence resource for A. sanguinea draft genome annotation, and will contribute to further molecular biology research on this harmful bloom algae.

High-resolution phylogenetic and population genetic analysis of microbial communities with RoC-ITS

Microbial communities are inter-connected systems of incredible complexity and dynamism that play crucial roles in health, energy, and the environment. To better understand microbial communities and how they respond to change, it is important to know which microbes are present and their relative abundances at the greatest taxonomic resolution possible. Here, we describe a novel protocol (RoC-ITS) that uses the single-molecule Nanopore sequencing platform to assay the composition of microbial communities at the subspecies designation. Using rolling-circle amplification, this methodology produces long-read sequences from a circular construct containing the complete 16S ribosomal gene and the neighboring internally transcribed spacer (ITS). These long reads can be used to generate a high-fidelity circular consensus sequence. Generally, the ribosomal 16S gene provides phylogenetic information down to the species-level, while the much less conserved ITS region contains strain-level information. When linked together, this combination of markers allows for the identification of individual ribosomal units within a specific organism and the assessment of their relative stoichiometry, as well as the ability to monitor subtle shifts in microbial community composition with a single generic assay. We applied RoC-ITS to an artificial microbial community that was also sequenced using the Illumina platform, to assess its accuracy in quantifying the relative abundance and identity of each species.

An Improved Model for Circular RNA Overexpression: Using the Actin Intron Reveals High Circularization Efficiency.

Traditionally, the group 1 intron of the T4 td gene is used to generate a foreign circular sequence. However, the T4 system has been shown to be fairly inefficient in expressing circular RNA (circRNA). Here, a new method is developed to express circular sequences with high circularization efficiency to strengthen the confidence for future circRNA functional studies. CircRNA expression plasmids, constructed with different lengths derived from the actin intron (15-nt, 30-nt, 60-nt, 100-nt, 180-nt) and T4 intron, are introduced into human and mouse cell lines 293T and B16. Junction detection and sequencing are used to determine successful circularization of introns and their expression efficiencies. An actin intron with a medium length (60-nt-100-nt) shows significantly increased efficiency of circularization, whereas intron-100-nt shows the best efficiency in most conditions. RNA pull-down assays are designed to precipitate the splicing factors that are bound to the introns and intron/exon junction. The precipitated proteins are analyzed by mass spectrometry (MS), aiming to identify the possible underlying mechanism behind the high circularization efficiency. This expression system has been validated using different circRNAs, and such method shows potential in contributing to the expanding field of circRNA studies.

Restriction Enzyme Maps of Circular Sequences of BBTV-DNA Components and Bioinformatics of Their Amino Acids

Restriction Enzyme Maps of Circular Sequences of BBTV-DNA Components and Bioinformatics of Their Amino Acids

Chromosome and Plasmid Sequences of Pantoea sp. Strain SOD02 Isolated from an Urban Freshwater Stream.

ABSTRACTAfter isolating Pantoea sp. strain SOD02 from an urban freshwater stream in Providence, RI, we used PacBio RSII data for de novo assembly and Illumina MiSeq data for polishing. This yielded complete circular sequences for a 4,227,027-bp chromosome with 54.7% GC and a 926,844-bp plasmid with 54.0% GC.