Aligned DNA Research Articles

Association of Acidotolerant Cyanobacteria to Microbial Mats below pH 1 in Acidic Mineral Precipitates in Río Tinto River in Spain.

This report describes acidic microbial mats containing cyanobacteria that are strongly associated to precipitated minerals in the source area of Río Tinto. Río Tinto (Huelva, Southwestern Spain) is an extreme acidic environment where iron and sulfur cycles play a fundamental role in sustaining the extremely low pH and the high concentration of heavy metals, while maintaining a high level of microbial diversity. These multi-layered mineral deposits are stable all year round and are characterized by a succession of thick greenish-blue and brownish layers mainly composed of natrojarosite. The temperature and absorbance above and below the mineral precipitates were followed and stable conditions were detected inside the mineral precipitates. Different methodologies, scanning and transmission electron microscopy, immunological detection, fluorescence in situ hybridization, and metagenomic analysis were used to describe the biodiversity existing in these microbial mats, demonstrating, for the first time, the existence of acid-tolerant cyanobacteria in a hyperacidic environment of below pH 1. Up to 0.46% of the classified sequences belong to cyanobacterial microorganisms, and 1.47% of the aligned DNA reads belong to the Cyanobacteria clade.

ORTHOSKIM: In silico sequence capture from genomic and transcriptomic libraries for phylogenomic and barcoding applications.

Low-coverage whole genome shotgun sequencing (or genome skimming) has emerged as a cost-effective method for acquiring genomic data in nonmodel organisms. This method provides sequence information on chloroplast genome (cpDNA), mitochondrial genome (mtDNA) and nuclear ribosomal regions (rDNA), which are over-represented within cells. However, numerous bioinformatic challenges remain to accurately and rapidly obtain such data in organisms with complex genomic structures and rearrangements, in particular for mtDNA in plants or for cpDNA in some plant families. Here we introduce the pipeline ORTHOSKIM, which performs in silico capture of targeted sequences from genomic and transcriptomic libraries without assembling whole organelle genomes. ORTHOSKIM proceeds in three steps: (i) global sequence assembly, (ii) mapping against reference sequences and (iii) target sequence extraction; importantly it also includes a range of quality control tests. Different modes are implemented to capture both coding and noncoding regions of cpDNA, mtDNA and rDNA sequences, along with predefined nuclear sequences (e.g., ultraconserved elements) or collections of single-copy orthologue genes. Moreover, aligned DNA matrices are produced for phylogenetic reconstructions, by performing multiple alignments of the captured sequences. While ORTHOSKIM is suitable for any eukaryote, a case study is presented here, using 114 genome-skimming libraries and four RNA sequencing libraries obtained for two plant families, Primulaceae and Ericaceae, the latter being a well-known problematic family for cpDNA assemblies. ORTHOSKIM recovered with high success rates cpDNA, mtDNA and rDNA sequences, well suited to accurately infer evolutionary relationships within these families. ORTHOSKIM is released under a GPL-3 licence and is available at: https://github.com/cpouchon/ORTHOSKIM.

Phylogenetic placement of a new Melanophilharmostes Paulian, 1968 pill scarab (Coleoptera: Hybosoridae: Ceratocanthinae) from Cameroon: molecular results decipher misleading morphology.

This study targets two specimens of a morphologically highly unusual pill scarab from Cameroon. The original morphology-inspired hypothesis that these beetles represent a new genus is refuted. Instead, a phylogenetic analysis of 90 terminals and 3231 aligned DNA positions linked both specimens with the Afrotropical genus Melanophilharmostes Paulian, 1968. Both Cameroonian specimens are herein described as Melanophilharmostes tuber Grebennikov, new species. Re-evaluation of the morphological characters of this species in light of molecular results corroborated this taxonomic decision by detecting all three morphological autapomorphies of the genus: the presence of setae and horseshoe-shaped microsculpture on the dorsal body surface, as well as the presence of the lateral carina on each elytron. Except for a single representative of the genus Melanophilharmostes placed within the genus Pseudopterorthochaetes Paulian, 1977 (interpreted as an analytical artifact), both genera are sister groups, in agreement with the pre-existing morphology-based hypothesis. Both genera, together with the Malagasy/Comorian genus Synarmostes Germar, 1843 and monotypic Balleriodes Grebennikov, 2021 from Tanzania, constitute the strongly supported Synarmostes group of genera.

Systematic Revision of the Arboreal Neotropical “Thorellii” Clade of Centruroides Marx, 1890, Bark Scorpions (Buthidae C.L. Koch, 1837) with Descriptions of Six New Species

The arboreal Neotropical “thorellii” clade of Centruroides Marx, 1890, bark scorpions (Buthidae C.L. Koch, 1837) is revised, using a novel approach to species delimitation. A phylogenetic analysis, based on 112 morphological characters and 1078 aligned DNA nucleotides from the mitochondrial Cytochrome c Oxidase Subunit I (COI) gene, provided the framework for placing singletons from geographically disparate localities (and often with suboptimal preservation) using COI minibarcodes, thereby enlarging the taxon sample for diagnosis and delimitation of morphological species. Six new species are described, tripling the known diversity in the clade to nine: Centruroides berstoni, sp. nov.; Centruroides catemacoensis, sp. nov.; Centruroides chanae, sp. nov.; Centruroides cuauhmapan, sp. nov.; Centruroides hamadryas, sp. nov.; Centruroides yucatanensis, sp. nov. Revised diagnoses are presented for Centruroides hoffmanni Armas, 1996, Centruroides rileyi Sissom, 1995, and Centruroides schmidti Sissom, 1995. Comparative images, a key and distribution maps for all species of the clade are provided, along with a summary of available data for their ecology.

Hypothesis Testing With Rank Conditions in Phylogenetics.

A phylogenetic model of sequence evolution for a set of n taxa is a collection of probability distributions on the 4n possible site patterns that may be observed in their aligned DNA sequences. For a four-taxon model, one can arrange the entries of these probability distributions into three flattening matrices that correspond to the three different unrooted leaf-labeled four-leaf trees, or quartet trees. The flattening matrix corresponding to the tree parameter of the model is known to satisfy certain rank conditions. Methods such as ErikSVD and SVDQuartets take advantage of this observation by applying singular value decomposition to flattening matrices consisting of empirical data. Each possible quartet is assigned an “SVD score” based on how close the flattening is to the set of matrices of the predicted rank. When choosing among possible quartets, the one with the lowest score is inferred to be the phylogeny of the four taxa under consideration. Since an n-leaf phylogenetic tree is determined by its quartets, this approach can be generalized to infer larger phylogenies. In this article, we explore using the SVD score as a test statistic to test whether phylogenetic data were generated by a particular quartet tree. To do so, we use several results to approximate the distribution of the SVD score and to give upper bounds on the p-value of the associated hypothesis tests. We also apply these hypothesis tests to simulated phylogenetic data and discuss the implications for interpreting SVD scores in rank-based inference methods.

An updated infra-familial classification of Sapindaceae based on targeted enrichment data.

PremiseThe economically important, cosmopolitan soapberry family (Sapindaceae) comprises ca. 1900 species in 144 genera. Since the seminal work of Radlkofer, several authors have attempted to overcome challenges presented by the family’s complex infra‐familial classification. With the advent of molecular systematics, revisions of the various proposed groupings have provided significant momentum, but we still lack a formal classification system rooted in an evolutionary framework.MethodsNuclear DNA sequence data were generated for 123 genera (86%) of Sapindaceae using target sequence capture with the Angiosperms353 universal probe set. HybPiper was used to produce aligned DNA matrices. Phylogenetic inferences were obtained using coalescence‐based and concatenated methods. The clades recovered are discussed in light of both benchmark studies to identify synapomorphies and distributional evidence to underpin an updated infra‐familial classification.Key ResultsCoalescence‐based and concatenated phylogenetic trees had identical topologies and node support, except for the placement of Melicoccus bijugatus Jacq. Twenty‐one clades were recovered, which serve as the basis for a revised infra‐familial classification.ConclusionsTwenty tribes are recognized in four subfamilies: two tribes in Hippocastanoideae, two in Dodonaeoideae, and 16 in Sapindoideae (no tribes are recognized in the monotypic subfamily Xanthoceratoideae). Within Sapindoideae, six new tribes are described: Blomieae Buerki & Callm.; Guindilieae Buerki, Callm. & Acev.‐Rodr.; Haplocoeleae Buerki & Callm.; Stadmanieae Buerki & Callm.; Tristiropsideae Buerki & Callm.; and Ungnadieae Buerki & Callm. This updated classification provides a backbone for further research and conservation efforts on this family.

DNA Curtains Shed Light on Complex Molecular Systems During Homologous Recombination.

Homologous recombination (HR) is important for the repair of double-stranded DNA breaks (DSBs) and stalled replication forks in all organisms. Defects in HR are closely associated with a loss of genome integrity and oncogenic transformation in human cells. HR involves coordinated actions of a complex set of proteins, many of which remain poorly understood. The key aspect of the research described here is a technology called "DNA curtains", a technique which allows for the assembly of aligned DNA molecules on the surface of a microfluidic sample chamber. They can then be visualized by total internal reflection fluorescence microscopy (TIRFM). DNA curtains was pioneered by our laboratory and allows for direct access to spatiotemporal information at millisecond time scales and nanometer scale resolution, which cannot be easily revealed through other methodologies. A major advantage of DNA curtains is that it simplifies the collection of statistically relevant data from single molecule experiments. This research continues to yield new insights into how cells regulate and preserve genome integrity.

Integrative systematics untangles the evolutionary history ofStenochrus(Schizomida: Hubbardiidae), a neglected junkyard genus of North American short-tailed whipscorpions

AbstractUntil recently, the Nearctic short-tailed whipscorpion genus, StenochrusChamberlin, 1922, included 27 species distributed primarily in Mexico, the USA and Central America. Morphological disparity among its species, associated with their adaptation to diverse habitats, raised the question as to whether Stenochrus was monophyletic. The phylogenetic relationships among short-tailed whipscorpions have only recently begun to be explored, and the monophyly of Stenochrus had never been tested. The present contribution provides the first phylogeny of Stenochrus and related genera, based on 61 morphological characters and 2991 aligned DNA nucleotides from two nuclear and two mitochondrial gene markers, for 73 terminal taxa. Separate and simultaneous analyses of the morphological and molecular data sets were conducted with Bayesian Inference, Maximum Likelihood, and parsimony with equal and implied weighting. Terminals represented only by morphological data (‘orphans’) were included in some analyses for evaluation of their phylogenetic positions. As previously defined, Stenochrus sensuReddell & Cokendolpher (1991, 1995) was consistently polyphyletic and comprised eight monophyletic clades, justifying its reclassification into eight genera including Heteroschizomus Rowland, 1973, revalidated from synonymy with Stenochrus by Monjaraz-Ruedas et al. (2019). Rowland & Reddell’s (1980)mexicanus and pecki species groups were consistently paraphyletic. Orphans grouped with the most morphologically similar taxa.

SPECIES TREE INFERENCE FROM GENOMIC SEQUENCES USING THE LOG-DET DISTANCE.

The log-det distance between two aligned DNA sequences was introduced as a tool for statistically consistent inference of a gene tree under simple non-mixture models of sequence evolution. Here we prove that the log-det distance, coupled with a distance-based tree construction method, also permits consistent inference of species trees under mixture models appropriate to aligned genomic-scale sequences data. Data may include sites from many genetic loci, which evolved on different gene trees due to incomplete lineage sorting on an ultrametric species tree, with different time-reversible substitution processes. The simplicity and speed of distance-based inference suggests log-det based methods should serve as benchmarks for judging more elaborate and computationally-intensive species trees inference methods.

BIIGA: Bioinformatics inspired image grouping approach

Initial work on image phylogeny used different approaches like the minimum spanning tree etc. The less investigated attempt is a bioinformatics-inspired approach for image phylogeny. The aim of this paper is to bridge this gap by generating image phylogeny with the concept of phylogenetic trees in bioinformatics. These trees were developed by using multiple aligned DNA sequences of original or multiple print-scan (MPS) degraded variants of watermarked (W) and non-watermarked (NW) images. Experimental results disclosed the viability of a proposed novel approach and effectively grouped original or MPS degraded variants of the W or NW images. The proposed method’s claims may revolutionise our knowledge of degraded (D) or non-degraded (ND) W and NW image grouping. It may direct to a new era of phylogenetic tree-based W or NW degraded or original images for developing the next generation of degraded (by MPS) W and NW grouping softwares. To the best of our knowledge, this is the first time such a method has been employed for image grouping. Our contributions are: (a) biologically-based image encoding to DNA letters (b) multiple sequence alignment (MSA) of DNA-encoded images (c) phylogenetic tree generation to group MPS-degraded W or NW images.

BACTOME-a reference database to explore the sequence- and gene expression-variation landscape of Pseudomonas aeruginosa clinical isolates.

Extensive use of next-generation sequencing (NGS) for pathogen profiling has the potential to transform our understanding of how genomic plasticity contributes to phenotypic versatility. However, the storage of large amounts of NGS data and visualization tools need to evolve to offer the scientific community fast and convenient access to these data. We introduce BACTOME as a database system that links aligned DNA- and RNA-sequencing reads of clinical Pseudomonas aeruginosa isolates with clinically relevant pathogen phenotypes. The database allows data extraction for any single isolate, gene or phenotype as well as data filtering and phenotypic grouping for specific research questions. With the integration of statistical tools we illustrate the usefulness of a relational database structure for the identification of phenotype–genotype correlations as an essential part of the discovery pipeline in genomic research. Furthermore, the database provides a compilation of DNA sequences and gene expression values of a plethora of clinical isolates to give a consensus DNA sequence and consensus gene expression signature. Deviations from the consensus thereby describe the genomic landscape and the transcriptional plasticity of the species P. aeruginosa. The database is available at https://bactome.helmholtz-hzi.de.

A molecular phylogeny of the Old World cypresses (Cupressus: Cupressaceae): evidence from nuclear and chloroplast DNA sequences

Studies of phylogenetic relationships among cypresses of the Old World (Cupressus; Cupressaceae) have been plagued by unresolved relationships, poor branch support, and conflict between data sets and methods of analysis. In this study, we combined 5.4 kb of aligned DNA sequence and 157 binary characters with previously published data in examining phylogenetic relationships among Cupressus species. Bayesian and parsimony analysis of the combined data or of the nuclear data alone always recovered three principal clades of Cupressus; however, tests of phylogenetic incongruence could not distinguish between competing relationships among the three principal Cupressus lineages. In contrast, incongruence tests often found statistically significant conflict between the nuclear and plastid data, particularly with respect to the placement of C. chengiana. Consistent with previous studies and prevailing taxonomic opinion, we find C. darjeelingensis more closely related to cypresses of the New World (Hesperocyparis). In contrast, we placed accessions of C. assamica and C. tonkinensis, two putatively Old World species suggested to be misidentified New World taxa by some authors, within well-supported Old World clades. Statistical analysis of genetic distances suggests instances in which taxa recognized as distinct species by some authors are identical or nearly so and may best be considered a single taxon. Conversely, we identify instances in which infraspecific taxa are more distantly related to one another than those traditionally recognized as distinct species. Factors confounding cypress taxonomies, including poor morphological differentiation, misidentification, and the use of accessions of questionable provenance, are discussed.

Atomic Force Microscopy Imaging Study of Aligning DNA by Dumbbell-like Au-Fe3O4 Magnetic Nanoparticles.

Studies on nucleic acid structure and interactions between nucleic acid and its binding molecules are of great importance for understanding and controlling many important biological processes. Atomic force microscopy (AFM) imaging is one of the most efficient methods to disclose the DNA structure and binding modes between DNA and DNA-binding molecules. Long-chain DNA tends to form a random coiled structure, which prevents direct AFM imaging observation of the subtle structure formed by DNA itself or protein binding. Aligning DNA from the random coiled state into the extended state is not only important for applications in DNA nanotechnology but also for elucidating the interaction mechanism between DNA and other molecules. Here, we developed an efficient method based on the magnetic field to align long-chain DNA on a silicon surface. We used AFM imaging to study the alignment of DNA at the single-molecule level, showing that DNA can be stretched and highly aligned by the manipulation of magnetic nanoparticles tethered to one end of DNA and that the aligned DNA can be imaged clearly by AFM. In the absence of the magnetic field, the aligned DNA can relax back to a random coiled state upon rinsing. Such alignment and relaxation can be repeated many times, which provides an efficient method for the manipulation of individual DNA molecules and the investigation of DNA and DNA-binding molecule interactions.

Single-Molecule Characterization of P53 on DNA using DNA Array “DNA Garden”

For tumor suppression, it is fundamental that p53 binds to target sites of DNA correctly. How does p53 search for and recognize the target sites among a huge DNA within a physiological time? Here, we developed a new method for producing the aligned DNA array, DNA garden (Bull. Chem. Soc. Jpn. 2017), and investigated the target search dynamics of p53 using a single-molecule fluorescence microscopy. p53 possessed two 1D-sliding modes along DNA, and the sliding distance was maintained under change of [Mg2+] and [Ca2+] (J. Mol. Biol. 2015). The modes and sliding distance were regulated by disordered C-terminal domain and linker (Biophys. J. 2017; Biochemistry 2017). Target recognition probability of p53 was 7% and was regulated by the activation and inactivation of p53 (J. Mol. Biol. 2016). Target recognition of p53 was achieved by 1D sliding along DNA, rather than 3D encounter. Furthermore, we developed high-speed single-molecule imaging system and applied to visualize the movement of p53 along DNA. Results showed that p53 can pass over obstacles on DNA by using intersegmental transfer or hopping/jumping. Overall, we will present how p53 behaves on DNA and how the observed dynamics of p53 are relevant to the biological functions.

Highly Aligned Plasmonic Gold Nanorods in a DNA Matrix

AbstractSince the Lycurgus Cup was made in the 4th century, metal nanoparticles have attracted much interest due to the characteristics of the plasmonic and metamaterials that show beautiful colors. Despite these fascinating properties, the practical use is limited because it is difficult to control the orientation of the plasmonic nanoparticles. Here, highly aligned plasmonic gold nanorods are obtained using self‐assembled DNA material. Simple mechanical shearing results in long‐range DNA–gold nanorod arrays which show parallel, perpendicular, and zigzag configurations due to the competition between the shear force and DNA elasticity. The resulting surface plasmonic resonance properties of the aligned DNA–gold nanorods film show highly polarization‐dependent behavior in a large area, which is critical for optical and photonic applications. This simple way to form anisotropic plasmonic films can be used for plasmonic nanoparticles in potential applications such as displays and sensors.

Hydration forces between aligned DNA helices undergoing B to A conformational change: In-situ X-ray fiber diffraction studies in a humidity and temperature controlled environment.

Hydration forces between DNA molecules in the A- and B-Form were studied using a newly developed technique enabling simultaneous in situ control of temperature and relative humidity. X-ray diffraction data were collected from oriented calf-thymus DNA fibers in the relative humidity range of 98%-70%, during which DNA undergoes the B- to A-form transition. Coexistence of both forms was observed over a finite humidity range at the transition. The change in DNA separation in response to variation in humidity, i.e. change of chemical potential, led to the derivation of a force-distance curve with a characteristic exponential decay constant of∼2Å for both A- and B-DNA. While previous osmotic stress measurements had yielded similar force-decay constants, they were limited to B-DNA with a surface separation (wall-to-wall distance) typically>5Å. The current investigation confirms that the hydration force remains dominant even in the dry A-DNA state and at surface separation down to∼1.5Å, within the first hydration shell. It is shown that the observed chemical potential difference between the A and B states could be attributed to the water layer inside the major and minor grooves of the A-DNA double helices, which can partially interpenetrate each other in the tightly packed A phase. The humidity-controlled X-ray diffraction method described here can be employed to perform direct force measurements on a broad range of biological structures such as membranes and filamentous protein networks.

Spontaneous formation of aligned DNA nanowires by capillarity-induced skin folding

Although DNA nanowires have proven useful as a template for fabricating functional nanomaterials and a platform for genetic analysis, their widespread use is still hindered because of limited control over the size, geometry, and alignment of the nanowires. Here, we document the capillarity-induced folding of an initially wrinkled surface and present an approach to the spontaneous formation of aligned DNA nanowires using a template whose surface morphology dynamically changes in response to liquid. In particular, we exploit the familiar wrinkling phenomenon that results from compression of a thin skin on a soft substrate. Once a droplet of liquid solution containing DNA molecules is placed on the wrinkled surface, the liquid from the droplet enters certain wrinkled channels. The capillary forces deform wrinkles containing liquid into sharp folds, whereas the neighboring empty wrinkles are stretched out. In this way, we obtain a periodic array of folded channels that contain liquid solution with DNA molecules. Such an approach serves as a template for the fabrication of arrays of straight or wrinkled DNA nanowires, where their characteristic scales are robustly tunable with the physical properties of liquid and the mechanical and geometrical properties of the elastic system.

Using Dendritic Heat Maps to Simultaneously Display Genotype Divergence with Phenotype Divergence.

The advancement of techniques to visualize and analyze large-scale sequencing datasets is an area of active research and is rooted in traditional techniques such as heat maps and dendrograms. We introduce dendritic heat maps that display heat map results over aligned DNA sequence clusters for a range of clustering cutoffs. Dendritic heat maps aid in visualizing the effects of group differences on clustering hierarchy and relative abundance of sampled sequences. Here, we artificially generate two separate datasets with simplified mutation and population growth procedures with GC content group separation to use as example phenotypes. In this work, we use the term phenotype to represent any feature by which groups can be separated. These sequences were clustered in a fractional identity range of 0.75 to 1.0 using agglomerative minimum-, maximum-, and average-linkage algorithms, as well as a divisive centroid-based algorithm. We demonstrate that dendritic heat maps give freedom to scrutinize specific clustering levels across a range of cutoffs, track changes in phenotype inequity across multiple levels of sequence clustering specificity, and easily visualize how deeply rooted changes in phenotype inequity are in a dataset. As genotypes diverge in sample populations, clusters are shown to break apart into smaller clusters at higher identity cutoff levels, similar to a dendrogram. Phenotype divergence, which is shown as a heat map of relative abundance bin response, may or may not follow genotype divergences. This joined view highlights the relationship between genotype and phenotype divergence for treatment groups. We discuss the minimum-, maximum-, average-, and centroid-linkage algorithm approaches to building dendritic heat maps and make a case for the divisive “top-down” centroid-based clustering methodology as being the best option visualize the effects of changing factors on clustering hierarchy and relative abundance.

A Robust Highly Aligned DNA Nanowire Array-Enabled Lithography for Graphene Nanoribbon Transistors.

Because of its excellent charge carrier mobility at the Dirac point, graphene possesses exceptional properties for high-performance devices. Of particular interest is the potential use of graphene nanoribbons or graphene nanomesh for field-effect transistors. Herein, highly aligned DNA nanowire arrays were crafted by flow-assisted self-assembly of a drop of DNA aqueous solution on a flat polymer substrate. Subsequently, they were exploited as "ink" and transfer-printed on chemical vapor deposited (CVD)-grown graphene substrate. The oriented DNA nanowires served as the lithographic resist for selective removal of graphene, forming highly aligned graphene nanoribbons. Intriguingly, these graphene nanoribbons can be readily produced over a large area (i.e., millimeter scale) with a high degree of feature-size controllability and a low level of defects, rendering the fabrication of flexible two terminal devices and field-effect transistors.

Alignment of Gold Nanoparticle-Decorated DNA Origami Nanotubes: Substrate Prepatterning versus Molecular Combing

DNA origami has become an established technique for designing well-defined nanostructures with any desired shape and for the controlled arrangement of functional nanostructures with few nanometer resolution. These unique features make DNA origami nanostructures promising candidates for use as scaffolds in nanoelectronics and nanophotonics device fabrication. Consequently, a number of studies have shown the precise organization of metallic nanoparticles on various DNA origami shapes. In this work, we fabricated large arrays of aligned DNA origami decorated with a high density of gold nanoparticles (AuNPs). To this end, we first demonstrate the high-yield assembly of high-density AuNP arrangements on DNA origami adsorbed to Si surfaces with few unbound background nanoparticles by carefully controlling the concentrations of MgCl2 and AuNPs in the hybridization buffer and the hybridization time. Then, we evaluate two methods, i.e., hybridization to prealigned DNA origami and molecular combing in a receding meniscus, with respect to their potential to yield large arrays of aligned AuNP-decorated DNA origami nanotubes. Because of the comparatively low MgCl2 concentration required for the efficient immobilization of the AuNPs, the prealigned DNA origami become mobile and displaced from their original positions, thereby decreasing the alignment yield. This increased mobility, on the other hand, makes the adsorbed origami susceptible to molecular combing, and a total alignment yield of 86% is obtained in this way.