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Several approaches have been proposed to sequence the genome of Vitis vinifera L. (grapevine), utilizing low-resolution second-generation platforms that lead to partial coverage and low-quality reads. Nevertheless, the characterization of the grapevine genetic resources could be better addressed through extensive and high-resolution genome sequencing. MinION is a third-generation sequencer preferred by many laboratories due to its relatively low cost, ease of use, and small size. To exploit MinION at its full potential, it requires high-quality DNA which is difficult to obtain in grapevine. Here we establish a workflow for DNA for MinION sequencing long reads from grapevine. This protocol was tested with undeveloped leaves, apex, helices, petioles, and 4th leaves samples. In addition, a circulomic size selection approach was tested to provide an unparalleled size of DNA reads. Our results suggest that longer reads of grapevine DNA associate with the use of fresh undeveloped tissue and adjusted conditions for the purification of cell nuclei. Short read elimination protocol increased N50 read length with a reasonable compromise for overall data yield. Our workflow presents a significant advancement for long-read quality DNA isolation for grapevine, providing also technical considerations for high-quality DNA preparation compatible with MinION from plants.

Tomato is cultivated in Greece for more than 200 years, even though is not native. Along with a favorable environment for the crop all the year round, Greece has become a major competitor in tomato production around Europe. However, there is an increasing demand to improve tomato crop. People have devoted a significant effort for crop improvement via phenotypic screening resulting in a large number of tomato ecotypes. An increasing demand to clarify the relationships among local tomato genotypes utilizing the most preferred molecular markers the Simple Sequence Repeats (SSR-markers) is the main objective of this study. Twenty-nine tomato ecotypes cultivated in Crete, Greece were genotyped utilizing eleven simple sequence repeat (SSR) along with the Structure analysis of the germplasm. A neighbor-joining dendrogram of the 29 ecotypes was produced. The statistical analysis proved that 58% of the genetic variation (p

Lefort and Roubelakis-Angelakis ([2000. The Greek Vitis Database: A multimedia web-backed genetic database for germplasm management of Vitis resources in Greece. Journal of Wine Research, 11(3), 23...

(Gaillon) Simonsen is famous among diatoms for producing a blue pigment. Genome sequencing of different strains of H. ostrearia led to the discovery of the complete genomes of two new bacilladnavir...

Background: There have been several attempts to sequence the genome of Vitis vinifera L. (grapevine), utilizing low-resolution second-generation platforms. Nevertheless, the characterization of the grapevine genetic resources and its adaptation to vulnerable conditions could be better addressed through extensive and high-resolution genome sequencing.MinION is a third-generation sequencer preferred by many laboratories due to its relatively low cost, ease of use and small size. Even though this long-read technology has been rapidly improving, to reach its full potential requires high-quality DNA.Results: Here we establish a workflow for DNA extraction suitable for MinION sequencing long reads from grapevine. This protocol was tested with leaf samples from different positions on annual growing branches of grapevine, Purified nuclei from fresh young leaves that led to high quality, long DNA fragments, suitable for long-read sequencing were successfully generated. It is evident that longer reads in grapevine associate with both fresh tissue and adjusted conditions used for nuclei purification.Conclusions: We propose that this workflow presents a significant advancement for long-read quality DNA isolation for grapevine and likely other plant species.

Molecular species identification plays a crucial role in archaeology and palaeontology, especially when diagnostic morphological characters are unavailable. Molecular markers have been used in forensic science to trace the geographic origin of wildlife products, such as ivory. So far, only a few studies have applied genetic methods to both identify the species and circumscribe the provenance of historic wildlife trade material. Here, by combining ancient DNA methods and genome skimming on a historical elephantid tooth found in southwestern Portugal, we aimed to identify its species, infer its placement in the elephantid phylogenetic tree, and triangulate its geographic origin. According to our results the specimen dates back to the eighteenth century CE and belongs to a female African forest elephant (non-hybrid Loxodonta cyclotis individual) geographically originated from west—west-central Africa, from areas where one of the four major mitochondrial clades of L. cyclotis is distributed. Historical evidence supports our inference, pointing out that the tooth should be considered as post-Medieval raw ivory trade material between West Africa and Portugal. Our study provides a comprehensive approach to study historical products and artefacts using archaeogenetics and contributes towards enlightening cultural and biological historical aspects of ivory trade in western Europe.

Technological advancements have revolutionized ancient and degraded DNA analysis, moving the field to the Next Generation Sequencing era. One of the advancements, the ancient DNA-oriented high-throughput library preparation methods, enabled the sequencing of more endogenous molecules. Although fairly optimized, both single- and double-stranded library preparation methods hold the potential for further improvement. Here, we test a series of modifications made at different steps of both single- and double-stranded library preparation methods. Given all the modifications tested, we found that two of them provide further benefits, including the use of Endonuclease VIII as a pre-treatment step before preparing single-stranded libraries and the use of a modified second adapter of the single stranded-libraries as an alternative option to enable sequencing of single stranded-libraries with the standard Illumina sequencing primer instead of the custom designed as described in the single stranded library preparation method. Furthermore, we propose uracil-DNA-glycosylase (UDG) could also be considered for both single- and double-stranded library preparation methods, although additional parameters should be taken into account depending on the sequencing strategy and the sample characteristics. Further modifications were also tested and although they were not advantageous, they could be considered as equivalent to the published options.

Organisms integrate information of current environmental stressors and can adjust themselves against harmful events that might occur in the future. The molecular processes that lead to such "anticipatory" behaviors, although of great interest, are mostly unexplored and the minimal genetic requirements for reconfiguring key signaling networks in order either to create or to strengthen such vital "anticipatory" capabilities is largely unknown. We identified new "anticipatory" phenotypes in yeast cells by evolving yeast strains that strongly associate a present modest stress with a future deadly one. Whole genome sequencing and classic genetic analysis revealed that two dominant negative ras2 alleles (ras2-K23N and ras2-G17C) displayed a strong "anticipatory" ability being highly resistant to oxidative stress, extremely thermotolerant and long lived only following an initial mild heat shock. We suggest that such "anticipatory" phenotypes can be easily evolved by a single point mutation in a key signaling protein, the Ras2 small GTPase, and we propose a molecular model describing how specific ras2 alleles, and not null ras2 mutants, or mutations in other components of the Ras/cAMP pathway, can enhance the "predictive ability" of cells for future lethal stressors.

Over the last 10 years, Next-Generation Sequencing (NGS) has become a powerful tool in clinical genetics and precision medicine. Techniques like Whole Genome Sequencing (WGS), Whole Exome Sequencing (WES) and Target Sequencing are commonly used for the elucidation of common and rare variants in mendelian and complex diseases. One of the most vital parts of NGS pipelines is the prioritization of annotated variants according to their clinical significance. During this process, a clinical geneticist is presented with annotation information from external databases for each of the thousands of potential variants. The vast amounts of data and the vague nature of existing guidelines for variant reporting, like ACMG (American College of Medical Genetics) can make this procedure very cumbersome and time consuming. Here we present the main computational challenges and existing solutions for this task. We also present Zazz, an online environment for variant annotation, query and exploration. Zazz can efficiently support the submission of complex and dynamically generated queries to hundreds of millions of variants each having hundreds of annotation fields. Zazz also leverages the capabilities of modern browsers to dynamically filter, explore and visualize multidimensional data.

The aim of this paper is to provide a snapshot of functional networks with central nodes (hubs) relying on the behavior of genes linked to a specific interaction network. Utilizing a ‘breast cancer signature’, a Bayesian approach is applied for the construction of gene interaction networks from different populations. We demonstrate that the hub genes of the differentiating network between cancer and control states can be regarded as potential markers for breast cancer. Furthermore, the differentiating subnetworks can be informative of the phenotype and provide new knowledge about the functional interactions and molecular pathways involved in breast cancer.

Hantaviruses are zoonotic viruses with a complex evolutionary history of virus-host coevolution and cross-species transmission. While hantaviruses have a broad reservoir host range, virus-host relationships were previously thought to be strict, with a single virus species infecting a single host species. Here, we describe Bruges virus, a novel hantavirus harbored by the European mole (Talpa europaea), which is the well-known host of Nova virus. Phylogenetic analyses of all three genomic segments showed tree topology inconsistencies, suggesting that Bruges virus has emerged from cross-species transmission and ancient reassortment events. A high number of co-infections with Bruges and Nova viruses was detected, but no evidence was found for reassortment between these two hantaviruses. These findings highlight the complexity of hantavirus evolution and the importance of further investigation of hantavirus-reservoir relationships. ispartof: Genome Biology and Evolution vol:10 issue:1 pages:45-55 ispartof: location:England status: published

In this paper, the authors present an assessment of the reliability of microarray experiments as well as their cross-laboratory/platform reproducibility rise as the major need. A critical challenge concerns the design of optimal universal reference rna (urr) samples to maximize detectable spots in two-color/channel microarray experiments, decrease the variability of microarray data, and finally ease the comparison between heterogeneous microarray datasets. Toward this target, the authors present an in-silico (binary) optimization process the solutions of which present optimal urr sample designs. Setting a cut-off threshold value over which a gene is considered as detectably expressed enables the process. Experimental results are quite encouraging and the related discussion highlights the suitability and flexibility of the approach.

Summarization: An increasing number of studies have profiled gene expressions in tumor specimens using distinct microarray plat forms and analysis techniques. One challenging task is to develop robust statistical models in order to integrate multi-platform findings. We compare some methodologies on the field with respect to estrogen receptor (ER) status, and focus on a unified-among platforms scale implemented by Shen et at. in 2004, which is based on a Bayesian mixture model. Under this scale, we study the ER intensity similarities between four breast cancer datasets derived from various platforms. We evaluate our results with an independent dataset in terms of ER sample classification, given the derived gene ER signatures of the integrated data. We found that integrated multi-platform gene signatures and fold-change variability similarities between different platform measurements can assist the statistical analysis of independent microarray datasets in terms of ER classification. Presented on: IEEE Transactions on Information Technology in Biomedicine

The detection of the biotin-streptavidin interaction is presented based on surface stress changes of functionalized Si membranes. During the biomolecular interaction, the surface stress of the functionalized membranes alters resulting in their deflection and finally in capacitance variations. The biological interaction on the sensors surface was verified after each experiment using fluorescent microscopy. Successful detection of 21nM of streptavidin using 3.55mM biotin probes is presented.

Laser Induced Forward Transfer (LIFT) technique is a direct-write method allowing the effective deposition of a wide range of materials with high spatial resolution. In this work, we present the direct printing of microarrays from a biotin solution by using a Nd:YAG laser (266nm wavelength, 4ns pulse duration). Standard materials of microelectronics such as Si and Low Temperature Oxide on Si (LTO/Si) were used as receiving substrates. For the LTO substrate, a detailed study for the optimum surface functionalization was carried out as it is a common passivation layer for biosensors. The laser beam parameters, such as the energy density and the beam size, were systematically varied in order to evaluate the effect on the transferred droplets. Furthermore, the energy density threshold for the ejection of the droplets was defined. Finally, the viability of the deposited droplets was demonstrated through the biotin-streptavidin reaction on silane coated LTO/Si substrates. This reaction was confirmed by fluorescence microscopy. These results have shown that the LIFT process can be used for biosensors and possibly for label-free detection by the combination of the method with capacitive sensors, sensors based on cantilevers or other sensors with electrical output.

The development of laser techniques for transferring biomolecules on solid surfaces in a controlled manner has attracted much attention during the last few years. Laser-induced forward transfer (LIFT) is a direct-write laser technique employed in printing micron-sized patterns or features of a wide range of materials in solid or liquid state, in ambient atmosphere and temperature or in vacuum. In this work, we use LIFT with nanosecond and femtosecond UV lasers for patterning the proteins Biotin, Avidin and Titin. In addition to the laser parameters, we also investigate the influence of the solution viscosity and the receiving surface wettability on the formed patterns. To confirm pattern functionality after transfer, we employ the strategy of patterning a coupling molecule with LIFT, immobilizing an active complementary protein on it and performing a fluorescence microscopy assay afterwards. Our results show that both nanosecond and femtosecond lasers are suitable tools for obtaining micron size protein patterns, with femtosecond laser pulses requiring a lower energy for transfer. We also show that the smallest patterns are formed just above the threshold energy for the thinnest target and that the pattern size increases with increasing surface wettability and decreases with solution viscosity.

The direct-writing technique laser-induced forward transfer has been employed for the micro-array printing of liquid solutions of the enzyme horseradish peroxidase and the protein Titin on nitrocellulose solid surfaces. The effect of two UV laser pulse lengths, femtosecond and nanosecond has been studied in relation with maintaining the activity of the transferred biomolecules. The quantification of the active biomolecules after transfer has been carried out using Bradford assay, quantitative colorimetric enzymatic assay and fluorescence techniques. Spectrophotometric measurements of the HRP and the Titin activity as well as chromatogenic and fluorescence assay studies have revealed a connection between the properties of the deposited, biologically active biomolecules, the experimental conditions and the target composition. The bioassays have shown that up to 78% of the biomolecules remained active after femtosecond laser transfer, while this value reduced to 54% after nanosecond laser transfer. The addition of glycerol in a percentage up to 70% in the solution to be transferred has contributed to the stabilization of the micro-array patterns and the increase of their resolution.

A comparative study of the effect of ultrashort (0.5 ps) and short (15 ns) pulses on the laser forward transfer of DNA molecules is presented in this paper. We use femtosecond laser pulses to directly print a wide range of biomaterials, in complicated patterns and structures. The ultrashort laser pulses reduce the thermal effects, thus allowing the effective deposition of sensitive biomaterials at high spatial resolution for micro-fabricating patterns. This direct laser printing process enables gentle and spatially selective transfer of biomaterials and facilitates application possibilities for the fabrication of biosensors and arrays for multi-analyte assays. Here, we present the direct micro-printing of biomaterials such as enzyme patterns by laser-induced forward transfer method using 500 fs laser pulses emitted at 248 nm. Furthermore, the dynamics of the process was investigated by stroboscopic schlieren imaging for time delays up to 3 μs following the laser irradiation pulse.

The precise fabrication of micro-dimensioned patterns of biomaterials by the laser microprinting technique using a sub-ps UV laser is described. An ultrashort UV laser has been used to transfer the biomaterial, with low angular divergence, and deposit it onto the substrate with minimum spread and high spatial resolution. The laser-transferred features of 100 μm ×100 μm size have been studied by means of scanning electron microscopy and scanning laser confocal fluorescence microscopy. The analysis of DNA and protein microarrays provides an excellent tool to expand our knowledge of genome functions.

The evaluation of a capacitive type biosensor array, consisting of a total of 256 biosensing elements, in the detection of single oligonucleotide mutations is presented. The biosensor takes advantage of surface stress changes during biological interactions and is able to translate them into a capacitive signal. The array is organized in a 16i?16 matrix of distinct biosensing elements thus allowing for the concurrent sensing of multiple biological targets. In this work the sensing elements of the array are spotted with three different oligonucleotides (CD8, CD17 and CD19) and their hybridization is detected using 36nM PCR. Moreover tests with CD19 revealed the ability of the biosensor to detect the hybridization of the oligo with sample concentrations of 36, 18 and 9nM.

Περίληψη: Osteoarthritis (OA) is a debilitating disease of the joints characterized by cartilage degradation. To date there is no available pharmacological treatment to inhibit disease progression neither is there a suitable biomarker for early disease prognosis. The aim of the present study was to investigate the molecular pathways involved between four identified OA susceptibility genes and their role in OA pathogenesis. Presented on: Osteoarthritis and Cartilage

The interconnection of heterogeneous data sources could provide a comprehensive picture of health parameters, thereby triggering an intervention by the medical staff upon detection of conditions that may lead to health deterioration, thus realizing preventive care. Supported Internet of Things technologies can be used to allow health related information to be locally aggregated and transmitted for remote monitoring and response. We present MyHealthAvatar (MHA), a personal digital health related collection bag, carried by individual citizens throughout their lifetime able to sustain in a meaningful manner all collected information. MHA acts as a unique companion continually following and empowering citizen and patients through a number of health related services. We describe the efforts on creating MHA patient-centered healthcare services for accessing, collecting and sharing long term multilevel personal health data through an integrated environment including: clinical data, genetic data, medical sensor data and devices, human behavior data and activity data for clinical data analysis, prediction and prevention for the individual citizen.

The generation of “virtual twins” of patients (Avatars) through integration of multiscale data gained from both the clinical profile of the patient and - omics tools, could create an appropriate environment for stratification of patients in fitting cohorts of “virtual populations”. Physiologically based pharmacokinetic & pharmacodynamic (PB/PK/PD) models as in silico clinical trial tools can estimate the PK/PD profiles in specific populations. In this work we discuss examples of how patient Avatars could be exploited in the context of in silico clinical trials and help in identifying novel biomarkers for personalized diagnosis. The PB/PK/PD models, neuroimaging and - omics data, may be fused together to further advance current decision making processes in clinical practice.

In this paper, the authors present an assessment of the reliability of microarray experiments as well as their cross-laboratory/platform reproducibility rise as the major need. A critical challenge concerns the design of optimal universal reference rna (urr) samples to maximize detectable spots in two-color/channel microarray experiments, decrease the variability of microarray data, and finally ease the comparison between heterogeneous microarray datasets. Toward this target, the authors present an in-silico (binary) optimization process the solutions of which present optimal urr sample designs. Setting a cut-off threshold value over which a gene is considered as detectably expressed enables the process. Experimental results are quite encouraging and the related discussion highlights the suitability and flexibility of the approach.