An Integrative Computational Approach for Prioritization of Genomic Variants

An essential step in the discovery of molecular mechanisms contributing to disease phenotypes and efficient experimental planning is the development of weighted hypotheses that estimate the functional effects of sequence variants discovered by high-throughput genomics. With the increasing specialization of the bioinformatics resources, creating analytical workflows that seamlessly integrate data and bioinformatics tools developed by multiple groups becomes inevitable. Here we present a case study of a use of the distributed analytical environment integrating four complementary specialized resources, namely the Lynx platform, VISTA RViewer, the Developmental Brain Disorders Database (DBDB), and the RaptorX server, for the identification of high-confidence candidate genes contributing to pathogenesis of spina bifida. The analysis resulted in prediction and validation of deleterious mutations in the SLC19A placental transporter in mothers of the affected children that causes narrowing of the outlet channel and therefore leads to the reduced folate permeation rate. The described approach also enabled correct identification of several genes, previously shown to contribute to pathogenesis of spina bifida, and suggestion of additional genes for experimental validations. The study demonstrates that the seamless integration of bioinformatics resources enables fast and efficient prioritization and characterization of genomic factors and molecular networks contributing to the phenotypes of interest.</p

A new mutant genetic resource for tomato crop improvement by TILLING technology

<p>Abstract</p> <p>Background</p> <p>In the last decade, the availability of gene sequences of many plant species, including tomato, has encouraged the development of strategies that do not rely on genetic transformation techniques (GMOs) for imparting desired traits in crops. One of these new emerging technology is TILLING (Targeting Induced Local Lesions In Genomes), a reverse genetics tool, which is proving to be very valuable in creating new traits in different crop species.</p> <p>Results</p> <p>To apply TILLING to tomato, a new mutant collection was generated in the genetic background of the processing tomato cultivar Red Setter by treating seeds with two different ethylemethane sulfonate doses (0.7% and 1%). An associated phenotype database, LycoTILL, was developed and a TILLING platform was also established. The interactive and evolving database is available online to the community for phenotypic alteration inquiries. To validate the Red Setter TILLING platform, induced point mutations were searched in 7 tomato genes with the mismatch-specific ENDO1 nuclease. In total 9.5 kb of tomato genome were screened and 66 nucleotide substitutions were identified. The overall mutation density was estimated and it resulted to be 1/322 kb and 1/574 kb for the 1% EMS and 0.7% EMS treatment respectively.</p> <p>Conclusions</p> <p>The mutation density estimated in our collection and its comparison with other TILLING populations demonstrate that the Red Setter genetic resource is suitable for use in high-throughput mutation discovery. The Red Setter TILLING platform is open to the research community and is publicly available via web for requesting mutation screening services.</p

Identification of Rice Transcription Factors Associated with Drought Tolerance Using the Ecotilling Method

The drought tolerance (DT) of plants is a complex quantitative trait. Under natural and artificial selection, drought tolerance represents the crop survival ability and production capacity under drought conditions (Luo, 2010). To understand the regulation mechanism of varied drought tolerance among rice genotypes, 95 diverse rice landraces or varieties were evaluated within a field screen facility based on the ‘line–source soil moisture gradient’, and their resistance varied from extremely resistant to sensitive. The method of Ecotype Targeting Induced Local Lesions in Genomes (Ecotilling) was used to analyze the diversity in the promoters of 24 transcription factor families. The bands separated by electrophoresis using Ecotilling were converted into molecular markers. STRUCTURE analysis revealed a value of K = 2, namely, the population with two subgroups (i.e., indica and japonica), which coincided very well with the UPGMA clusters (NTSYS-pc software) using distance-based analysis and InDel markers. Then the association analysis between the promoter diversity of these transcription factors and the DT index/level of each variety was performed. The results showed that three genes were associated with the DT index and that five genes were associated with the DT level. The sequences of these associated genes are complex and variable, especially at approximately 1000 bp upstream of the transcription initiation sites. The study illuminated that association analysis aimed at Ecotilling diversity of natural groups could facilitate the isolation of rice genes related to complex quantitative traits

Rapid identification of causal mutations in tomato EMS populations via mapping-by-sequencing

The tomato is the model species of choice for fleshy fruit development and for the Solanaceae family. Ethyl methanesulfonate (EMS) mutants of tomato have already proven their utility for analysis of gene function in plants, leading to improved breeding stocks and superior tomato varieties. However, until recently, the identification of causal mutations that underlie particular phenotypes has been a very lengthy task that many laboratories could not afford because of spatial and technical limitations. Here, we describe a simple protocol for identifying causal mutations in tomato using a mapping-by-sequencing strategy. Plants displaying phenotypes of interest are first isolated by screening an EMS mutant collection generated in the miniature cultivar Micro-Tom. A recombinant F2 population is then produced by crossing the mutant with a wild-type (WT; non-mutagenized) genotype, and F2 segregants displaying the same phenotype are subsequently pooled. Finally, whole-genome sequencing and analysis of allele distributions in the pools allow for the identification of the causal mutation. The whole process, from the isolation of the tomato mutant to the identification of the causal mutation, takes 6-12 months. This strategy overcomes many previous limitations, is simple to use and can be applied in most laboratories with limited facilities for plant culture and genotyping

Types of polymerisation units and their intensity output in private dental clinics of twin cities in eastern province, KSA; a pilot study

الملخص: أهداف البحث: تُعتبر الحشوات التركيبية ذات الأساس الراتنجي المعالجة بالضؤء هي الخيار المفضل لترميم الأسنان. ينبغي على وحدات المعالجة الضوئية المستخدمة للأسنان أن توفر طاقة ضوئية كافية لضمان خواص ميكانيكية جيدة وثبات الأبعاد والتوافق الحيوي مع الحشوات التركيبية ذات الأساس الراتنجي. وكان الهدف من هذه الدراسة هو تحديد أنواع وحدات المعالجة الضوئية وكثافة إخراجها الضوئي في عيادات الأسنان الخاصة. طرق البحث: تم استحداث نموذج لتسجيل المعلومات المتعلقة بنوع مصابيح المعالجة وكثافة إخراجها الضوئي. تم تقييم ما مجموعه ٤٠٠ جهاز علاج باستخدام مقياس إشعاع رقمي في ٥٨ عيادة أسنان خاصة. وأُخذت ثلاث قراءات منفصلة مدة كل منها ١٠ ثوانٍ لكل جهاز وتم احتساب المتوسط. واعتُبرت كثافة إخراج أقل من ٣٠٠ ميجاواط لكل سم مربع بالنسبة لوحدات الهالوجين غير مرضية، بينما اعتبرت القراءة غير مرضية لوحدات الصمام الثنائي الباعث للضوء إذا كانت أقل من ٦٠٠ ميجاواط لكل سم مربع. النتائج: من بين ٤٠٠ مصباح علاجي، كان هناك ٣٥٤ مصباح صمام ثنائي باعث للضوء و٤٦ وحدة مصباح هالوجين. واعتبر ما مجموعه ١٣٪ من المصابيح غير مرضية. كانت الكثافة الضوئيه في ١٢.٤٪ من وحدات الصمام الثنائي الباعث للضوء أقل من ٦٠٠ ميجاواط لكل سم مربع، في حين كانت الكثافة الضوئيه في ١٧.٣٪ من وحدات الهالوجين أقل من ٣٠٠ ميجاواط لكل سم مربع. الاستنتاجات: أظهرت دراسة وحدات المعالجة الضوئية أن هنالك توجه في اتجاه وحدات العلاج الضوئي من نوع الصمام الثنائي الباعث للضوء في عيادات الاسنان الخاصة، في حين أن قيمة الكثافة الضوئية المتوسطة الصادرة من وحدات العلاج الضوئي من نوع الصمام الثنائي الباعث للضوء كانت أعلى من تلك الصادرة من وحدات الهالوجين. ويعتبر مقياس الإشعاع، بشكل عام، أداة جيدة لتقييم الكثافة الضوئية الصادرة من وحدات العلاج الضوئي. Abstract: Objectives: Light-cured resin-based composites (RBCs) are the preferred option to restore teeth. Dental light-curing units (LCUs) should deliver adequate light energy to ensure good mechanical properties, dimensional stability, and biocompatibility of the RBC. The aim of this study was to determine the types of LCUs and their intensity output in private dental clinics. Methods: A form was developed to record information related to the type of curing lights and their intensity output. A total of 400 curing devices were evaluated using a digital radiometer in 58 private dental clinics. For each device, three separate 10-s readings were taken and the average was calculated. For quartz tungsten halogen (QTH) units, a light intensity below 300 mW/cm2 was considered unsatisfactory, whereas for light-emitting diode (LED) units, a reading below 600 mW/cm2 was considered unsatisfactory. Results: Out of 400 curing lights, 354 were LEDs and 46 were QTH units. A total of 13% of the lights were considered unsatisfactory. Of the LED units, 12.4% had a light intensity of less than 600 mW/cm2, whereas QTH had 17.3% units with an intensity of less than 300 mW/cm2. Conclusion: The frequency of LCUs showed a trend towards LED units in private dental clinics, whereas the mean intensity value from the LED was higher than that from QTH units. Overall, the radiometer is a good tool to assess the intensity output of LCUs. الكلمات المفتاحية: الصمام الثنائي الباعث للضوء, الهالوجين الكوارتزي التنغستني, مقياس الإشعاع, الحشوات التركيبية الراتنجية, Keywords: Light-emitting diode, Quartz tungsten halogen, Radiometer, Resin composit

An Integrative Computational Approach for Prioritization of Genomic Variants

<div><p>An essential step in the discovery of molecular mechanisms contributing to disease phenotypes and efficient experimental planning is the development of weighted hypotheses that estimate the functional effects of sequence variants discovered by high-throughput genomics. With the increasing specialization of the bioinformatics resources, creating analytical workflows that seamlessly integrate data and bioinformatics tools developed by multiple groups becomes inevitable. Here we present a case study of a use of the distributed analytical environment integrating four complementary specialized resources, namely the Lynx platform, VISTA RViewer, the Developmental Brain Disorders Database (DBDB), and the RaptorX server, for the identification of high-confidence candidate genes contributing to pathogenesis of spina bifida. The analysis resulted in prediction and validation of deleterious mutations in the SLC19A placental transporter in mothers of the affected children that causes narrowing of the outlet channel and therefore leads to the reduced folate permeation rate. The described approach also enabled correct identification of several genes, previously shown to contribute to pathogenesis of spina bifida, and suggestion of additional genes for experimental validations. The study demonstrates that the seamless integration of bioinformatics resources enables fast and efficient prioritization and characterization of genomic factors and molecular networks contributing to the phenotypes of interest.</p></div