Exome Sequencing from Nanogram Amounts of Starting DNA: Comparing Three Approaches

Hybridization-based target enrichment protocols require relatively large starting amounts of genomic DNA, which is not always available. Here, we tested three approaches to pre-capture library preparation starting from 10 ng of genomic DNA: (i and ii) whole-genome amplification of DNA samples with REPLI-g (Qiagen) and GenomePlex (Sigma) kits followed by standard library preparation, and (iii) library construction with a low input oriented ThruPLEX kit (Rubicon Genomics). Exome capture with Agilent SureSelect^XT2 Human AllExon v4+UTRs capture probes, and HiSeq2000 sequencing were performed for test libraries along with the control library prepared from 1 µg of starting DNA. Tested protocols were characterized in terms of mapping efficiency, enrichment ratio, coverage of the target region, and reliability of SNP genotyping. REPLI-g- and ThruPLEX-FD-based protocols seem to be adequate solutions for exome sequencing of low input samples.     

Knocking out multigene redundancies via cycles of sexual assortment and fluorescence selection

Phenotypes that might otherwise reveal a gene's function can be obscured by genes with overlapping function. This phenomenon is best known within gene families, in which an important shared function may only be revealed by mutating all family members. Here we describe the 'green monster' technology that enables precise deletion of many genes. In this method, a population of deletion strains with each deletion marked by an inducible green fluorescent protein reporter gene, is subjected to repeated rounds of mating, meiosis and flow-cytometric enrichment. This results in the aggregation of multiple deletion loci in single cells. The green monster strategy is potentially applicable to assembling other engineered alterations in any species with sex or alternative means of allelic assortment. To test the technology, we generated a single broadly drug-sensitive strain of Saccharomyces cerevisiae bearing precise deletions of all 16 ATP-binding cassette transporters within clades associated with multidrug resistance.     

Atomistic simulations of materials for optical chemical sensors: DFT-D calculations of molecular interactions between gas-phase analyte molecules and simple substrate models

The structures of complexes of some small molecules (formaldehyde, acetaldehyde, ammonia, methylamine, methanol, ethanol, acetone, benzene, acetonitrile, ethyl acetate, chloroform, and tetrahydrofuran, considered as possible analytes) with ethylbenzene and silanol (C₆H₅C₂H₅ and SiH₃OH, considered as models of polystyrene and silica gel substrates) and with acridine (C₁₃H₉N, considered as a model of an indicator dye molecule of the acridine series) and the corresponding interaction energies have been calculated using the DFT-D approximation. The PBE exchange-correlation potential was used in the calculations. The structures of complexes between the analyte and the substrate were determined by optimizing their ground-state geometry using the SVP split-valence double-zeta plus polarization basis set. The complex formation energies were refined by single-point calculations at the calculated equilibrium geometries using the sufficiently large triple-zeta TZVPP basis set. The calculated interaction energies are used to assess the possibility of using dyes of the acridine series adsorbed on a polystyrene or silica substrate for detecting the small molecules listed above.     

Oxirapentyns B–D produced by a marine sediment-derived fungus Isaria felina (DC.) Fr

Three new highly oxygenated chromene derivatives, oxirapentyns B–D (1–3) and one known oxirapentyn A (4) were isolated from the lipophilic extract of the marine-derived fungus Isaria felina KMM 4639. The structures of compounds 1–3 were determined based on spectroscopic methods. The absolute configuration of oxirapentyn B (1) as 2R, 4S, 5S, 6S, 7R, 8S, 9S was established by a combination of modified Mosher's method, X-ray analysis, and NOESY data. Oxirapentyn A (4) showed weak cytotoxicity against SK-Mel-5, SK-Mel-28 human malignant melanoma, and T-47D human breast cancer cell lines.     

High level accumulation of gamma linolenic acid (C18:3Δ6.9,12 cis) in transgenic safflower (Carthamus tinctorius) seeds

Gamma linolenic acid (GLA; C18:3Δ6,9,12 cis), also known as γ-Linolenic acid, is an important essential fatty acid precursor for the synthesis of very long chain polyunsaturated fatty acids and important pathways involved in human health. GLA is synthesized from linoleic acid (LA; C18:2Δ9,12 cis) by endoplasmic reticulum associated Δ6-desaturase activity. Currently sources of GLA are limited to a small number of plant species with poor agronomic properties, and therefore an economical and abundant commercial source of GLA in an existing crop is highly desirable. To this end, the seed oil of a high LA cultivated species of safflower (Carthamus tinctorius) was modified by transformation with Δ6-desaturase from Saprolegnia diclina resulting in levels exceeding 70% (v/v) of GLA. Levels around 50% (v/v) of GLA in seed oil was achieved when Δ12-/Δ6-desaturases from Mortierella alpina was over-expressed in safflower cultivars with either a high LA or high oleic (OA; C18:1Δ9 cis) background. The differences in the overall levels of GLA suggest the accumulation of the novel fatty acid was not limited by a lack of incorporation into the triacylgylcerol backbone (>66% GLA achieved), or correlated with gene dosage (GLA levels independent of gene copy number), but rather reflected the differences in Δ6-desaturase activity from the two sources. To date, these represent the highest accumulation levels of a newly introduced fatty acid in a transgenic crop. Events from these studies have been propagated and recently received FDA approval for commercialization as Sonova?400.     

Ultrastructural characterization and comparative phylogenetic analysis of new microsporidia from Siberian mosquitoes: evidence for coevolution and host switching

A survey of mosquito larvae infected with microsporidia was conducted from 2005 to 2008 in the Tomsk, Kemerovo and Novosibirsk regions of western Siberia, Russia. Twenty-one morphologically and genetically unique species of microsporidia were isolated from nine species of Anopheles, Aedes, Culex and Ochlerotatus mosquitoes including: (1) 14 proposed new species of Amblyospora (A. bakcharia, A. baritia, A. bogashovia, A. chulymia, A. hristinia, A. jurginia, A. kazankia, A. mavlukevia, A. mocrushinia, A. modestium, A. salairia, A. severinia, A. shegaria, and A. timirasia); (2) a newly proposed genus and species, Novothelohaniaovalae and; (3) six species of Amblyospora (A. flavescens, A. kolarovi, A. rugosa), Parathelohania (P. divulgata and P. tomski) and Trichoctosporea (T. pygopellita) from which gene sequences had not been previously obtained. Detailed ultrastructure of meiospores revealed unique cytological features associated with the length, arrangement and ratio of broad to narrow coils of the polar filament, comparative thickness of the exospore and endospore, and overall size of each species reaffirming their value in distinguishing taxonomic relationships. SSU rDNA sequences obtained from each species of microsporidia were unique when compared with GenBank entries. Phylogenetic trees constructed by Maximum Parsimony, Maximum Likelihood and Neighbor Joining analyses yielded similar topologies with a high degree of congruence between parasite and host at the generic level. Species that parasitize Aedes/Ochlerotatus and Culex mosquitoes segregate into distinct monophyletic groupings mirroring their host phylogeny, while species from Anopheles mosquitoes group as a sister clade basal to the entire group of mosquito-parasitic microsporidia as their Anopheles hosts cluster as a sister clade to the entire group of culicine mosquitoes. This provides strong evidence for host-parasite coevolution by descent at the generic level and limited host lineage switching between unrelated taxa. Among parasites of Aedes/Ochlerotatus and Anopheles mosquitoes, we found several instances where a single mosquito species serves as a host for two or more related species of microsporidia, an observation consistent with host switching and independent parasite speciation. Among the microsporidian parasites of Culex mosquitoes, we found only one parasite per host indicating a higher degree of host specificity and less host switching among parasites of this genus. Findings suggest a degree of host-parasite co-speciation with host switching occurring occasionally when the "normal" host is unavailable in the aquatic ecosystem. Frequency of host switching seems to be occurring in proportion to host relatedness and does not cross generic boundaries in this system.     

A panel of microsatellite loci for population studies of Sakhalin taimen Parahucho perryi (Brevoort)

Using DNA samples of Sakhalin taimen and a set of microsatellite loci, earlier reported for other salmonid fishes (Salmonidae), successful cross-species amplification was performed. A total of 56 Sakhalin taimen (Parahucho perryi) samples from the Daga, Nabil, Poronai, and Agnevo rivers (Sakhalin Island) were examined at 36 microsatellite loci, most of which were described for other species and first tested in taimen. Among the 21 loci first tested in taimen, two loci produced no amplification products. The remaining 19 loci were successfully amplified (for some loci, new primers were generated). Thirteen of these loci were monomorphic, while six loci were polymorphic and used in further population genetic analysis. In addition, with the purpose of modification of the allele sizes and optimization of the research technique, new primers for the already known 12 loci of Sakhalin taimen were designed. Three more loci were included in analysis without changes. As a result, a joint panel consisting of 21 polymorphic microsatellite markers was suggested for analysis of Sakhalin taimen. This panel was tested with four population samples from the rivers of Sakhalin Island.The results showed that this panel of markers could be used in detailed population studies for evaluation of the level of genetic differentiation, inbreeding, and migrations in Sakhalin taimen, an endangered species with a fragmented range. Using this approach in further studies will make it possible to isolate basic populations, which is necessary for conservation of this rare species.     

Occurrence of emerald ash borer (Coleoptera: Buprestidae) and biotic factors affecting its immature stages in the Russian Far East

Field surveys were conducted from 2008 to 2011 in the Khabarovsk and Vladivostok regions of Russia to investigate the occurrence of emerald ash borer, Agrilus planipennis Fairmaire, and mortality factors affecting its immature stages. We found emerald ash borer infesting both introduced North American green ash (Fraxinus pennsylvanica Marshall) and native oriental ashes (F. mandshurica Rupr. and F. rhynchophylla Hance) in both regions. Emerald ash borer densities (larvae/m(2) of phloem area) were markedly higher on green ash (11.3-76.7 in the Khabarovsk area and 77-245 in the Vladivostok area) than on artificially stressed Manchurian ash (2.2) or Oriental ash (10-59). Mortality of emerald ash borer larvae caused by different biotic factors (woodpecker predation, host plant resistance and/or undetermined diseases, and parasitism) varied with date, site, and ash species. In general, predation of emerald ash borer larvae by woodpeckers was low. While low rates (3-27%) of emerald ash borer larval mortality were caused by undetermined biotic factors on green ash between 2009 and 2011, higher rates (26-95%) of emerald ash borer larval mortality were caused by putative plant resistance in Oriental ash species in both regions. Little (<1%) parasitism of emerald ash borer larvae was observed in Khabarovsk; however, three hymenopteran parasitoids (Spathius sp., Atanycolus nigriventris Vojnovskaja-Krieger, and Tetrastichus planipennisi Yang) were observed attacking third - fourth instars of emerald ash borer in the Vladivostok area, parasitizing 0-8.3% of emerald ash borer larvae infesting Oriental ash trees and 7.3-62.7% of those on green ash trees (primarily by Spathius sp.) in two of the three study sites. Relevance of these findings to the classical biological control of emerald ash borer in newly invaded regions is discussed.