Circular Sequence Research Articles

Identification and characterization of a novel geminivirus with a monopartite genome infecting apple trees.

A novel circular DNA virus sequence has been identified through next-generation sequencing and in silico assembly of small RNAs of 21-24 nt from an apple tree grown in China. The virus genome was cloned using two independent approaches and sequenced. With a size of 2932 nt, it showed the same genomic structure and conserved origin of replication reported for members of the family Geminiviridae. However, the low nucleotide and amino acid sequence identity with known geminiviruses indicated that it was a novel virus, for which the provisional name apple geminivirus (AGV) is proposed. Rolling circle amplification followed by RFLP analyses indicated that AGV was a virus with a monopartite DNA genome. This result was in line with bioassays showing that the cloned viral genome was infectious in several herbaceous plants (Nicotiana bethamiana, Nicotiana glutinosa and Solanum lycopersicum), thus confirming it was complete and biologically active, although no symptoms were observed in these experimental hosts. AGV genome structure and phylogenetic analyses did not support the inclusion of this novel species in any of the established genera in the family Geminiviridae. A survey of 165 apple trees grown in four Chinese provinces showed a prevalence of 7.2% for AGV, confirming its presence in several cultivars and geographical areas in China, although no obvious relationship between virus infection and specific symptoms was found.

The complete mitochondrial genome of the killifish Orestias sp. (Cyprinodontiformes, Cyprinodontidae) from the high Andean range

The killifish genus Orestias is endemic to freshwater ecosystems in the High Andes of Peru, Bolivia and Chile. Phylogenetic and phylogeographic studies with partial mitochondrial genes have failed to resolve relationship among species, needing more comprehensive approaches. In this study, we described the complete mitochondrial genome of Orestias sp. from Lirima, northern Chile, with the aim to provide useful data for phylogenetic purposes and species delimitation. The mitochondrial genome was assembled with 2.6 million of reads obtained through an Ion Torrent (chip 318) sequencer. The circular sequence of 16,617 bp showed the following nucleotide composition: A, 26.7%, C, 27.1%, G, 17.0%, and T, 29.2%. Gene composition and structure were similar to other fish sequences available, and comprised 13 protein-coding genes, 12S and 16S rRNA, 22 tRNA genes, and a control region.

Deletion of DNA sequences of using a polymerase chain reaction based approach

We developed a simple, rapid and reliable method to delete DNA fragments in plasmids using a polymerase chain reaction based amplification of the circular DNA sequence that excludes the fragment to be deleted. The primers are designed to contain a non-complementary 5' sequence consisting of a restriction enzyme target sequence. Following PCR amplification, the plasmid is digested with Dpn I to eliminate the template DNA, with the chosen restriction enzyme, and ligated. The only limitation is the selection of the restriction enzyme target sequence that must not be present in the original plasmid. The method is straightforward in its execution and success relies on a meticulous primer design that permits us obtain 100% of transformants containing the desired mutation. The extraordinary simplicity of the method makes it a valuable tool to generate DNA deletions in plasmids and to study the effects of those deletions in protein function.

Computing halfspace depth contours based on the idea of a circular sequence

This paper presents a new efficient algorithm for exactly computing the halfspace depth contours based on the idea of a circular sequence. Unlike the existing methods, the proposed algorithm segments the unit sphere directly relying on the permutations that correspond to the projections of observations onto some unit directions, without having to use the technique of parametric programming. Some data examples are also provided to illustrate the performance of the proposed algorithm.

The complete mitochondrial genome of the rocky reef fish Cheilodactylus variegatus Valenciennes, 1833 (Teleostei: Cheilodactylidae)

Cheilodactylus variegatus is a common benthivorous marine fish inhabiting in rocky subtidal habitats in the eastern south Pacific coast of Chile and Peru. However, its biology and ecology are relatively understudied and its taxonomic assignment has been debated recently. The complete mitochondrial genome was assembled de novo and mapped to a reference using 5.97 million of reads obtained through Ion Torrent next generation sequencing, resulting in a circular sequence of 16,652 bp in length. Gene composition and arrangement comprised to that reported for most fishes and contained the typical structure of 2 rRNAs, 13 protein-coding genes, 22 tRNAs and 1 non-coding region. This mitogenome provides a valuable resource for studies of fish molecular systematics, phylogeography and population genetics.

The complete chloroplast genome of colchicine plants (Colchicum autumnale L. and Gloriosa superba L.) and its application for identifying the genus.

The complete chloroplast genome of two colchicine medicinal plants is reported for the first time. Deletion of ycf 15 gene occurred only in Colchicum but not in Gloriosa and suggests this as a potential marker for delineating the two species. Colchicum autumnale L. and Gloriosa superba L. are well-known sources of colchicine, a type of alkaloid and an ancient anti-inflammatory drug used to prevent gout. Accordingly, this alkaloid has been used as a chemical marker for identifying the expanded Colchicaceae family. In the present study, we report the complete chloroplast genome (cpDNA) sequence of two colchicine medicinal plants (G. superba and C. autumnale) that belong to the tribe Colchiceae of the Colchicaceae family. In C. autumnale, the circular double-stranded cpDNA sequence of 156,462 bp consists of two inverted repeat (IR) regions of 27,741 bp each, a large single-copy region (LSC) of 84,246 bp, and a small single-copy region (SSC) of 16,734 bp. The cpDNA sequence of G. superba is longer than that of C. autumnale (157,924 bp), which consists of two IRs (28,063 bp), an SSC (16,786 bp), and an LSC (85,012 bp). Significant structural differences between them were observed in the ycf15 gene. ycf15 gene was absent from C. autumnale cpDNA and affected the length of the chloroplast genome between the species. Furthermore, this gene loss event was specific to the expanded genus of Colchicum sensu Vinnersten and Manning. Therefore, this gene may be an effective and powerful molecular marker for identifying the Colchicum genus within the family.

Helical Disruptions in Small Loops of DNA

The thermodynamical stability of DNA minicircles is investigated by means of path integral techniques. Hydrogen bonds between base pairs on complementary strands can be broken by thermal fluctuations and temporary fluctuational openings along the double helix are essential to biological functions such as transcription and replication of the genetic information. Helix unwinding and bubble formation patterns are computed in circular sequences with variable radius in order to analyze the interplay between molecule size and appearance of helical disruptions. The latter are found in minicircles with < 100 base pairs and appear as a strategy to soften the stress due to the bending and torsion of the helix.

Genome sequence determination and metagenomic characterization of a Dehalococcoides mixed culture grown on cis-1,2-dichloroethene

A Dehalococcoides-containing bacterial consortium that performed dechlorination of 0.20 mM cis-1,2-dichloroethene to ethene in 14 days was obtained from the sediment mud of the lotus field. To obtain detailed information of the consortium, the metagenome was analyzed using the short-read next-generation sequencer SOLiD 3. Matching the obtained sequence tags with the reference genome sequences indicated that the Dehalococcoides sp. in the consortium was highly homologous to Dehalococcoides mccartyi CBDB1 and BAV1. Sequence comparison with the reference sequence constructed from 16S rRNA gene sequences in a public database showed the presence of Sedimentibacter, Sulfurospirillum, Clostridium, Desulfovibrio, Parabacteroides, Alistipes, Eubacterium, Peptostreptococcus and Proteocatella in addition to Dehalococcoides sp. After further enrichment, the members of the consortium were narrowed down to almost three species. Finally, the full-length circular genome sequence of the Dehalococcoides sp. in the consortium, D. mccartyi IBARAKI, was determined by analyzing the metagenome with the single-molecule DNA sequencer PacBio RS. The accuracy of the sequence was confirmed by matching it to the tag sequences obtained by SOLiD 3. The genome is 1,451,062 nt and the number of CDS is 1566, which includes 3 rRNA genes and 47 tRNA genes. There exist twenty-eight RDase genes that are accompanied by the genes for anchor proteins. The genome exhibits significant sequence identity with other Dehalococcoides spp. throughout the genome, but there exists significant difference in the distribution RDase genes. The combination of a short-read next-generation DNA sequencer and a long-read single-molecule DNA sequencer gives detailed information of a bacterial consortium.

The Tight Coupling and Non-Linear Relationship between the Macroscopic Electrical and Optical Concomitants of Electrochemical CNS Waves Reflect the Non-Linear Dynamics of Neural Glial Propagation

In isolated chick retina, the visualization of electrochemical self-organized patterns is possible due to the presence of macroscopic intrinsic optical signals (IOSs). Isolated circular waves, standing patterns, and self-sustained sequences of spirals are all easily obtained using an IOS approach. In this paper we present the tight coupling and non-linear relationship between optical and electrical wave concomitants, and potassium-induced whole tissue excitability changes. Elementary statistical methods and time series analyses were applied to two sets of data: 1) solitary circular retinal spreading depression waves, and 2) tissue response to exogenous potassium fast pulses. The results were interpreted from the point of view of non-linear thermodynamical concepts and volume phase transitions in polyanionic gels according to the Tasaki action potential model. From these and previous results, it is clear that the glial network and extracellular matrix contribute to the propagation and emergence of these patterns.

The genome of the nucleopolyhedrosis-causing virus from Tipula oleracea sheds new light on the Nudiviridae family.

A large double-stranded DNA (dsDNA) virus that produces occlusion bodies, typical of baculoviruses, has been described to infect crane fly larvae of the genus Tipula (Diptera, Tipulidae). Because of a lack of genomic data, this virus has remained unclassified. Electron microscopy of an archival virus isolated from Tipula oleracea, T. oleracea nudivirus (ToNV), showed irregularly shaped occlusion bodies measuring from 2 to 5 μm in length and 2 μm in middiameter, filled with rod-shape virions containing single nucleocapsids within a bilayer envelope. Whole-genome amplification and Roche 454 sequencing revealed a complete circular genome sequence of 145.7 kb, containing five direct repeat regions. We predicted 131 open reading frames, including a homolog of the polyhedrin gene encoding the major occlusion body protein of T. paludosa nucleopolyhedrovirus (NPV). BLAST searches demonstrated that ToNV had 21 of the 37 baculovirus core genes but shared 52 genes with nudiviruses (NVs). Phylogenomic analyses indicated that ToNV clearly belongs to the Nudiviridae family but should probably be assigned to a new genus. Among nudiviruses, ToNV was most closely related to the Penaeus monodon NV and Heliothis zea NV clade but distantly related to Drosophila innubia NV, the other nudivirus infecting a Diptera. Lastly, ToNV was found to be most closely related to the nuvidirus ancestor of bracoviruses. This was also reflected in terms of gene content, as ToNV was the only known exogenous virus harboring homologs of the Cc50C22.6 and 27b (Cc50C22.7) genes found in the nudiviral genomic cluster involved in bracovirus particle production. The Nudiviridae is a family of arthropod dsDNA viruses from which striking cases of endogenization have been reported (i.e., symbiotic bracoviruses deriving from a nudivirus and the endogenous nudivirus of the brown planthopper). Although related to baculoviruses, relatively little is known about the genomic diversity of exogenous nudiviruses. Here, we characterized, morphologically and genetically, an archival sample of the Tipula oleracea nudivirus (ToNV), which has the particularity of forming occlusion bodies. Comparative genomic and phylogenomic analyses showed ToNV to be to date the closest known relative of the exogenous ancestor of bracoviruses and that ToNV should be assigned to a new genus. Moreover, we revised the homology relationships of nudiviral genes and identified a new set of 32 core genes for the Nudiviridae, of which 21 were also baculovirus core genes. These findings provide important insights into the evolutionary history of large arthropod dsDNA viruses.

The complete genome sequence of Candidatus Liberibacter americanus, a bacterium associated with Citrus Huanglongbing in Brazil

We used PFGE followed by CsCl bisbenzymide centrifugation to obtain sufficient DNA for pyrosequencing of the Ca. Liberibacter americanus (Lam) strain “Sao Paulo” genome. The complete circular genomic DNA sequence of Lam is 1,195,201 bp, with an average GC content of 31.12%, somewhat lower than other Liberibacters. There are 1,056 predicted Lam genes, with 1,002 encoding proteins, 9 encoding rRNA genes and 45 encoding tRNAs. The overall gene organization and structure of the Lam genome is more similar to Lso than to Las. There are 951 genes common to Lam, Lso and Las, 27 genes found in Lam and Lso but not Las, and only 8 genes common to Lam and Las but not found in Lso. Many pseudogenes or truncated genes were found among the unique genes of all 3 species. As with Las, two prophage were confirmed in Lam, with SP2 being 39,941 bp and SP1 being 16,398 bp in size; as in Las, the one that appears to replicate as an excision plasmid prophage carries putative lysogenic conversion genes, specifically peroxidases and a Type Vc secreted adhesin. These predicted peroxidases and adhesin were found in both Las and Lam, but appeared fragmentary or degenerated in Lso, indicating their potential for citrus host range determination. Liberibacter genomes seem to be under selective pressure to reduce GC% content and to lose unimportant genes. Although Lam have an outer membrane, most of the genes required for biosynthesis of lipopolysaccharide, which can trigger ROS production, are missing from the Lam genome.

Complete genome determination and analysis of Acholeplasma oculi strain 19L, highlighting the loss of basic genetic features in the Acholeplasmataceae.

BackgroundAcholeplasma oculi belongs to the Acholeplasmataceae family, comprising the genera Acholeplasma and ‘Candidatus Phytoplasma’. Acholeplasmas are ubiquitous saprophytic bacteria. Several isolates are derived from plants or animals, whereas phytoplasmas are characterised as intracellular parasitic pathogens of plant phloem and depend on insect vectors for their spread. The complete genome sequences for eight strains of this family have been resolved so far, all of which were determined depending on clone-based sequencing.ResultsThe A. oculi strain 19L chromosome was sequenced using two independent approaches. The first approach comprised sequencing by synthesis (Illumina) in combination with Sanger sequencing, while single molecule real time sequencing (PacBio) was used in the second. The genome was determined to be 1,587,120 bp in size. Sequencing by synthesis resulted in six large genome fragments, while the single molecule real time sequencing approach yielded one circular chromosome sequence. High-quality sequences were obtained by both strategies differing in six positions, which are interpreted as reliable variations present in the culture population. Our genome analysis revealed 1,471 protein-coding genes and highlighted the absence of the F1FO-type Na+ ATPase system and GroEL/ES chaperone. Comparison of the four available Acholeplasma sequences revealed a core-genome encoding 703 proteins and a pan-genome of 2,867 proteins.ConclusionsThe application of two state-of-the-art sequencing technologies highlights the potential of single molecule real time sequencing for complete genome determination. Comparative genome analyses revealed that the process of losing particular basic genetic features during genome reduction occurs in both genera, as indicated for several phytoplasma strains and at least A. oculi. The loss of the F1FO-type Na+ ATPase system may separate Acholeplasmataceae from other Mollicutes, while the loss of those genes encoding the chaperone GroEL/ES is not a rare exception in this bacterial class.Electronic supplementary materialThe online version of this article (doi:10.1186/1471-2164-15-931) contains supplementary material, which is available to authorized users.

Complete genome sequence of the mitochondrial DNA of the sparkling enope squid, Watasenia scintillans

The sparkling enope squid, Watasenia scintillans, is a deep-sea mollusk inhabiting the western part of the Pacific Ocean. It has the peculiar ability to illuminate its body without the involvement of other organisms. In this study, we extracted the brain DNA from a single squid female caught in the Japan Sea and determined the complete genome sequence of its mitochondrial DNA using the Illumina sequencing platform. The circular sequence is 20,089 bp in length. Using the next-generation sequencing data, we also estimated the mean copy number of mitochondria per cell in the brain to be 108 by comparing the depths of the read data in the nuclear and mitochondrial genomes. The haploid genome size was calculated to be 4.78 Gb. Six heteroplasmy sites were also identified, together with their allele frequencies, in this individual. Our methodology is shown to be useful in mitochondrion-related studies.

Chaotic image encryption based on circular substitution box and key stream buffer

A new image encryption algorithm based on spatiotemporal chaotic system is proposed, in which the circular S-box and the key stream buffer are introduced to increase the security. This algorithm is comprised of a substitution process and a diffusion process. In the substitution process, the S-box is considered as a circular sequence with a head pointer, and each image pixel is replaced with an element of S-box according to both the pixel value and the head pointer, while the head pointer varies with the previous substituted pixel. In the diffusion process, the key stream buffer is used to cache the random numbers generated by the chaotic system, and each image pixel is then enciphered by incorporating the previous cipher pixel and a random number dependently chosen from the key stream buffer. A series of experiments and security analysis results demonstrate that this new encryption algorithm is highly secure and more efficient for most of the real image encryption practices.

An efficient chaotic image encryption based on alternate circular S-boxes

A novel and efficient image encryption algorithm based on chaos and multiple S-boxes is proposed in this paper, in which a set of S-boxes is initially constructed using a chaotic system, and each of the S-boxes is considered as a circular sequence with a head pointer. For each image pixel, an S-box is chosen from the set of S-boxes and used to substitute for the plain pixel to get a cipher pixel, and then the chosen S-box is updated by moving its head pointer forward according to the cipher pixel and a random number. In order to increase the plaintext sensitivity of encryption, the substitution processes are performed in forward direction and backward direction, respectively. This scheme not only offers the high security by employing two directional substitutions and using the different S-boxes for each pixel but also achieves high encryption speed by constructing only a few S-boxes and updating the S-box dynamically and easily. The performance of the proposed algorithm is evaluated using a variety of analysis. Experimental results show that the proposed image encryption algorithm is secure and efficient.

Hundreds of Circular Novel Plasmids and DNA Elements Identified in a Rat Cecum Metamobilome

Metagenomic approaches are widespread in microbiological research, but so far, the knowledge on extrachromosomal DNA diversity and composition has largely remained dependant on cultivating host organisms. Even with the emergence of metagenomics, complete circular sequences are rarely identified, and have required manual curation. We propose a robust in silico procedure for identifying complete small plasmids in metagenomic datasets from whole genome shotgun sequencing. From one very pure and exhaustively sequenced metamobilome from rat cecum, we identified a total of 616 circular sequences, 160 of which were carrying a gene with plasmid replication domain. Further homology analyses indicated that the majority of these plasmid sequences are novel. We confirmed the circularity of the complete plasmid candidates using an inverse-type PCR approach on a subset of sequences with 95% success, confirming the existence and length of discrete sequences.The implication of these findings is a broadened understanding of the traits of circular elements in nature and the possibility of massive data mining in existing metagenomic datasets to discover novel pools of complete plasmids thus vastly expanding the current plasmid database.

The utility of PacBio circular consensus sequencing for characterizing complex gene families in non-model organisms.

BackgroundMolecular characterization of highly diverse gene families can be time consuming, expensive, and difficult, especially when considering the potential for relatively large numbers of paralogs and/or pseudogenes. Here we investigate the utility of Pacific Biosciences single molecule real-time (SMRT) circular consensus sequencing (CCS) as an alternative to traditional cloning and Sanger sequencing PCR amplicons for gene family characterization. We target vomeronasal gene receptors, one of the most diverse gene families in mammals, with the goal of better understanding intra-specific V1R diversity of the gray mouse lemur (Microcebus murinus). Our study compares intragenomic variation for two V1R subfamilies found in the mouse lemur. Specifically, we compare gene copy variation within and between two individuals of M. murinus as characterized by different methods for nucleotide sequencing. By including the same individual animal from which the M. murinus draft genome was derived, we are able to cross-validate gene copy estimates from Sanger sequencing versus CCS methods.ResultsWe generated 34,088 high quality circular consensus sequences of two diverse V1R subfamilies (here referred to as V1RI and V1RIX) from two individuals of Microcebus murinus. Using a minimum threshold of 7× coverage, we recovered approximately 90% of V1RI sequences previously identified in the draft M. murinus genome (59% being identical at all nucleotide positions). When low coverage sequences were considered (i.e. < 7× coverage) 100% of V1RI sequences identified in the draft genome were recovered. At least 13 putatively novel V1R loci were also identified using CCS technology.ConclusionsRecent upgrades to the Pacific Biosciences RS instrument have improved the CCS technology and offer an alternative to traditional sequencing approaches. Our results suggest that the Microcebus murinus V1R repertoire has been underestimated in the draft genome. In addition to providing an improved understanding of V1R diversity in the mouse lemur, this study demonstrates the utility of CCS technology for characterizing complex regions of the genome. We anticipate that long-read sequencing technologies such as PacBio SMRT will allow for the assembly of multigene family clusters and serve to more accurately characterize patterns of gene copy variation in large gene families, thus revealing novel micro-evolutionary patterns within non-model organisms.Electronic supplementary materialThe online version of this article (doi:10.1186/1471-2164-15-720) contains supplementary material, which is available to authorized users.

Microsatellite Marker Discovery using Single Molecule Real-Time Circular Consensus Sequencing on the Pacific Biosciences RS

Microsatellite sequences are important markers for population genetics studies. In the past, the development of adequate microsatellite primers has been cumbersome. However with the advent of next-generation sequencing technologies, marker identification in genomes of non-model species has been greatly simplified. Here we describe microsatellite discovery on a Pacific Biosciences single molecule real-time sequencer. For the Greater White-fronted Goose (Anser albifrons), we identified 316 microsatellite loci in a single genome shotgun sequencing experiment. We found that the capability of handling large insert sizes and high quality circular consensus sequences provides an advantage over short read technologies for primer design. Combined with a straightforward amplification-free library preparation, PacBio sequencing is an economically viable alternative for microsatellite discovery and subsequent PCR primer design.

On the profust reliabilities of consecutive k-out-of-n systems

System reliability concept is a very popular topic among scientific research areas. Systems may be classified based on their structures such as linear and circular, based on their evaluation characteristics such as fuzzy and nonfuzzy states and based on working principles such as F and G systems. Linear and circular system distinction is originated from ordering difference of components. System and/or component states can be evaluated as fuzzy and nonfuzzy. If system and component states are both evaluated as nonfuzzy, then the reliability of such a system is considered as conventional reliability. On the contrary, if system or component states are evaluated as fuzzy, then reliability of such a system is considered as fuzzy reliability. While the working principle is defined with respect to the number of failed components for an F system, it is defined with respect to the number of working components for a G system. A consecutive k-out-of- n:F system composes of a sequence of n ordered components such that the system is failed if and only if at least k consecutive components in the system are failed. Similarly, consecutive k-out-of- n:G system composes of a sequence of n ordered components such that the system works if and only if at least k consecutive components in the system work. In this study, we research the profust (states of components are evaluated as nonfuzzy and state of system is evaluated as fuzzy) reliabilities of consecutive k-out-of- n:F and G systems considering both linear and circular sequence of n components. The reliabilities of these systems are evaluated for independent and identically distributed components. Easy-to-use formulas for the reliabilities of these systems are obtained using the distribution of some run statistics. In order to use these formulas, the working probability of a component at any time is sufficient. Some numerical results and figures are also presented to acquire some information about the reliabilities of related systems and to compare performance differences between conventional and profust reliabilities of systems considered in this study.

OrganellarGenomeDRAW—a suite of tools for generating physical maps of plastid and mitochondrial genomes and visualizing expression data sets

Mitochondria and plastids (chloroplasts) are cell organelles of endosymbiotic origin that possess their own genetic information. Most organellar DNAs map as circular double-stranded genomes. Across the eukaryotic kingdom, organellar genomes display great size variation, ranging from ∼15 to 20 kb (the size of the mitochondrial genome in most animals) to >10 Mb (the size of the mitochondrial genome in some lineages of flowering plants). We have developed OrganellarGenomeDraw (OGDRAW), a suite of software tools that enable users to create high-quality visual representations of both circular and linear annotated genome sequences provided as GenBank files or accession numbers. Although all types of DNA sequences are accepted as input, the software has been specifically optimized to properly depict features of organellar genomes. A recent extension facilitates the plotting of quantitative gene expression data, such as transcript or protein abundance data, directly onto the genome map. OGDRAW has already become widely used and is available as a free web tool (http://ogdraw.mpimp-golm.mpg.de/). The core processing components can be downloaded as a Perl module, thus also allowing for convenient integration into custom processing pipelines.