Allele-specific expression assays using Solexa

<p>Abstract</p> <p>Background</p> <p>Allele-specific expression (ASE) assays can be used to identify <it>cis</it>, <it>trans</it>, and <it>cis</it>-by-<it>trans </it>regulatory variation. Understanding the source of expression variation has important implications for disease susceptibility, phenotypic diversity, and adaptation. While ASE is commonly measured via relative fluorescence at a SNP, next generation sequencing provides an opportunity to measure ASE in an accurate and high-throughput manner using read counts.</p> <p>Results</p> <p>We introduce a Solexa-based method to perform large numbers of ASE assays using only a single lane of a Solexa flowcell. In brief, transcripts of interest, which contain a known SNP, are PCR enriched and barcoded to enable multiplexing. Then high-throughput sequencing is used to estimate allele-specific expression using sequencing counts. To validate this method, we measured the allelic bias in a dilution series and found high correlations between measured and expected values (r>0.9, p < 0.001). We applied this method to a set of 5 genes in a <it>Drosophila simulans </it>parental mix, F1 and introgression and found that for these genes the majority of expression divergence can be explained by <it>cis</it>-regulatory variation.</p> <p>Conclusion</p> <p>We present a new method with the capacity to measure ASE for large numbers of assays using as little as one lane of a Solexa flowcell. This will be a valuable technique for molecular and population genetic studies, as well as for verification of genome-wide data sets.</p

Local analysis of porous silicon structure fabricated by nontraditional approach

© Published under licence by IOP Publishing Ltd. A comparison of experimental electron backscattering diffraction patterns for porous Si formed by ion implantation and thermal annealing is presented. For this purposes Ag-ion implantation into monocrystalline c-Si substrates at energy of 30 keV with dose of 1.5×1017 ion/cm2 was carried out. Surface nanoporous Si structures were studied by scanning electron microscope imaging and electron backscattering diffraction. Amorphization of Si after implantation and recrystallization of porous Si after annealing is observed. Ion implantation is suggested to be effective technique for a formation of nanoporous semiconductor layers, which could be easily combined with the crystalline substrate matrix for various applications

The markers of autoimmune inflammation in applied cardiovascular surgery

The negative influence of the autoimmune inflammation on vascular pathomorphology hasn t studied enough. There isn’t algorithm using markers of significant autoimmune inflammation of vascular wall in cardiovascular surgery. The study comprises 94 consecutive patients who were operated on for coronary revascularization. We divided the study into 2 groups (basic and control). Including criteria for basic group was hs-CRP (C-reactive protein) level in serum higher than 6 mg/l. as marker of vascular autoimmune inflammation. Control group included patients without vascular inflammation and with hs-CRP ievel in serum below 6 mg/l. Groups were compared on 28 clinical, laboratory and instrumental criteria. We found significant (p<0,005) clinical markers of intravascular inflammation in 26 (55%) patients from basic group, this fact made us to appoint immunotherapy in this patient before coronary revascularization. The use of immunotherapy in patient with intravascular inflammation before operation reduced the number of postoperative tromboembolic complications on 33% and mortality — on 23,8% (p<0,005). We suggest that our markers of autoimmune vascular inflammation are effective in clinical practice. Preoperative immunotherapy can prevent postoperative tromboembolic complications in patients undergoing coronary revascularization.Отрицательное влияние аутоиммунного воспалительного компонента на течение сосудистого патоморфоза в настоящее время мало изучено. До настоящего времени не разработан алгоритм с использованием маркеров аутоиммунного клинически значимого воспаления сосудистой стенки в сердечно-сосудистой хирургии. Проведено углубленное обследование 94 больных с ишемической болезнью сердца, направленных на хирургическую реваскуляризацию миокарда. Из всех пациентов было сформировано 2 равные группы (основная и контрольная). Критерием включения в основную группу был уровень С-реактивного белка (СРВ) в плазме крови выше 6 мг/л, как маркера сосудистого аутоиммунного воспаления. Контрольная группа включала пациентов без признаков воспаления сосудистого русла, с уровнем СРБ<6 мг/л. Сравнение полученных групп производилось по 28 клинико-лабораторным и инструментальным критериям. Во время исследования у пациентов из основной группы были выявлены статистически значимые (р<0,05) клинические маркеры внутрисосудистого воспаления, что потребовало проведение предоперационной иммуннокоррегирующей терапии у 26 (55%) пациентов основной группы. Проведенная иммунокоррекция позволила снизить процент послеоперационных тромботических осложнений на 33%, а смертность на 23,8% (р<0,05). Полученные результаты свидетельствуют об эффективности использованных нами маркеров внутрисосудистого воспаления в клинической практике, а проведение иммуннокоррекции позволило избежать послеоперационных тромботических осложнений

Genetic diversity, population structure, and genetic correlation with climatic variation in chickpea (Cicer arietinum) landraces from Pakistan

Chickpea (Cicer arietinum L.) production in arid regions, such as those predominant in Pakistan, faces immense challenges of drought and heat stress. Addressing these challenges is made more difficult by the lack of genetic and phenotypic characterization of available cultivated varieties and breeding materials. Genotyping-by-sequencing offers a rapid and cost-effective means to identify genome-wide nucleotide variation in crop germplasm. When combined with extended crop phenotypes deduced from climatic variation at sites of collection, the data can predict which portions of genetic variation might have roles in climate resilience. Here we use 8113 single nucleotide polymorphism markers to determine genetic variation and compare population structure within a previously uncharacterized collection of 77 landraces and 5 elite cultivars, currently grown in situ on farms throughout the chickpea growing regions of Pakistan. The compiled landraces span a striking aridity gradient into the Thal Desert of the Punjab. Despite low levels of variation across the collection and limited genetic structure, we found some differentiation between accessions from arid, semiarid, irrigated, and coastal areas. In a subset of 232 markers, we found evidence of differentiation along gradients of elevation and isothermality. Our results highlight the utility of exploring large germplasm collections for nucleotide variation associated with environmental extremes, and the use of such data to nominate germplasm accessions with the potential to improve crop drought tolerance and other environmental traits

Fine-Scale Mapping of Natural Variation in Fly Fecundity Identifies Neuronal Domain of Expression and Function of an Aquaporin

To gain insight into the molecular genetic basis of standing variation in fitness related traits, we identify a novel factor that regulates the molecular and physiological basis of natural variation in female Drosophila melanogaster fecundity. Genetic variation in female fecundity in flies derived from a wild orchard population is heritable and largely independent of other measured life history traits. We map a portion of this variation to a single QTL and then use deficiency mapping to further refine this QTL to 5 candidate genes. Ubiquitous expression of RNAi against only one of these genes, an aquaporin encoded by Drip, reduces fecundity. Within our mapping population Drip mRNA level in the head, but not other tissues, is positively correlated with fecundity. We localize Drip expression to a small population of corazonin producing neurons located in the dorsolateral posterior compartments of the protocerebrum. Expression of Drip–RNAi using both the pan-neuronal ELAV-Gal4 and the Crz-Gal4 drivers reduces fecundity. Low-fecundity RILs have decreased Crz expression and increased expression of pale, the enzyme encoding the rate-limiting step in the production of dopamine, a modulator of insect life histories. Taken together these data suggest that natural variation in Drip expression in the corazonin producing neurons contributes to standing variation in fitness by altering the concentration of two neurohormones

Natural Variation in Decision-Making Behavior in Drosophila melanogaster

There has been considerable recent interest in using Drosophila melanogaster to investigate the molecular basis of decision-making behavior. Deciding where to place eggs is likely one of the most important decisions for a female fly, as eggs are vulnerable and larvae have limited motility. Here, we show that many natural genotypes of D. melanogaster prefer to lay eggs near nutritious substrate, rather than in nutritious substrate. These preferences are highly polymorphic in both degree and direction, with considerable heritability (0.488) and evolvability

Contrasting Patterns of Sequence Evolution at the Functionally Redundant bric à brac Paralogs in Drosophila melanogaster

Genes with overlapping expression and function may gradually diverge despite retaining some common functions. To test whether such genes show distinct patterns of molecular evolution within species, we examined sequence variation at the bric à brac (bab) locus of Drosophila melanogaster. This locus is composed of two anciently duplicated paralogs, bab1 and bab2, which are involved in patterning the adult abdomen, legs, and ovaries. We have sequenced the 148 kb genomic region spanning the bab1 and bab2 genes from 94 inbred lines of D. melanogaster sampled from a single location. Two non-coding regions, one in each paralog, appear to be under selection. The strongest evidence of directional selection is found in a region of bab2 that has no known functional role. The other region is located in the bab1 paralog and is known to contain a cis-regulatory element that controls sex-specific abdominal pigmentation. The coding region of bab1 appears to be under stronger functional constraint than the bab2 coding sequences. Thus, the two paralogs are evolving under different selective regimes in the same natural population, illuminating the different evolutionary trajectories of partially redundant duplicate genes

Generalized shrinkage F-like statistics for testing an interaction term in gene expression analysis in the presence of heteroscedasticity

<p>Abstract</p> <p>Background</p> <p>Many analyses of gene expression data involve hypothesis tests of an interaction term between two fixed effects, typically tested using a residual variance. In expression studies, the issue of variance heteroscedasticity has received much attention, and previous work has focused on either between-gene or within-gene heteroscedasticity. However, in a single experiment, heteroscedasticity may exist both within and between genes. Here we develop flexible shrinkage error estimators considering both between-gene and within-gene heteroscedasticity and use them to construct <it>F</it>-like test statistics for testing interactions, with cutoff values obtained by permutation. These permutation tests are complicated, and several permutation tests are investigated here.</p> <p>Results</p> <p>Our proposed test statistics are compared with other existing shrinkage-type test statistics through extensive simulation studies and a real data example. The results show that the choice of permutation procedures has dramatically more influence on detection power than the choice of <it>F </it>or <it>F</it>-like test statistics. When both types of gene heteroscedasticity exist, our proposed test statistics can control preselected type-I errors and are more powerful. Raw data permutation is not valid in this setting. Whether unrestricted or restricted residual permutation should be used depends on the specific type of test statistic.</p> <p>Conclusions</p> <p>The <it>F</it>-like test statistic that uses the proposed flexible shrinkage error estimator considering both types of gene heteroscedasticity and unrestricted residual permutation can provide a statistically valid and powerful test. Therefore, we recommended that it should always applied in the analysis of real gene expression data analysis to test an interaction term.</p