Genetic differentiation of hypothalamus parentally biased transcripts in populations of the house mouse implicate the Prader-Willi syndrome imprinted region as a possible source of behavioral divergence.

Parentally biased expression of transcripts (genomic imprinting) in adult tissues, including the brain, can influence and possibly drive the evolution of behavioral traits. We have previously found that paternally determined cues are involved in population-specific mate choice decisions between two populations of the Western house mouse (Mus musculus domesticus). Here, we ask whether this could be mediated by genomically imprinted transcripts that are subject to fast differentiation between these populations. We focus on three organs that are of special relevance for mate choice and behavior: The vomeronasal organ (VNO), the hypothalamus, and the liver. To first identify candidate transcripts at a genome-wide scale, we used reciprocal crosses between M. m. domesticus and M. m. musculus inbred strains and RNA sequencing of the respective tissues. Using a false discovery cutoff derived from mock reciprocal cross comparisons, we find a total of 66 imprinted transcripts, 13 of which have previously not been described as imprinted. The largest number of imprinted transcripts were found in the hypothalamus; fewer were found in the VNO, and the least were found in the liver. To assess molecular differentiation and imprinting in the wild-derived M. m. domesticus populations, we sequenced the RNA of the hypothalamus from individuals of these populations. This confirmed the presence of the above identified transcripts also in wild populations and allowed us to search for those that show a high genetic differentiation between these populations. Our results identify the Ube3a-Snrpn imprinted region on chromosome 7 as a region that encompasses the largest number of previously not described transcripts with paternal expression bias, several of which are at the same time highly differentiated. For four of these, we confirmed their imprinting status via single nucleotide polymorphism-specific pyrosequencing assays with RNA from reciprocal crosses. In addition, we find the paternally expressed Peg13 transcript within the Trappc9 gene region on chromosome 15 to be highly differentiated. Interestingly, both regions have been implicated in Prader-Willi nervous system disorder phenotypes in humans. We suggest that these genomically imprinted regions are candidates for influencing the population-specific mate-choice in mice.

From next-generation sequencing alignments to accurate comparison and validation of single-nucleotide variants: the pibase software.

Scientists working with single-nucleotide variants (SNVs), inferred by next-generation sequencing software, often need further information regarding true variants, artifacts and sequence coverage gaps. In clinical diagnostics, e.g. SNVs must usually be validated by visual inspection or several independent SNV-callers. We here demonstrate that 0.5-60% of relevant SNVs might not be detected due to coverage gaps, or might be misidentified. Even low error rates can overwhelm the true biological signal, especially in clinical diagnostics, in research comparing healthy with affected cells, in archaeogenetic dating or in forensics. For these reasons, we have developed a package called pibase, which is applicable to diploid and haploid genome, exome or targeted enrichment data. pibase extracts details on nucleotides from alignment files at user-specified coordinates and identifies reproducible genotypes, if present. In test cases pibase identifies genotypes at 99.98% specificity, 10-fold better than other tools. pibase also provides pair-wise comparisons between healthy and affected cells using nucleotide signals (10-fold more accurately than a genotype-based approach, as we show in our case study of monozygotic twins). This comparison tool also solves the problem of detecting allelic imbalance within heterozygous SNVs in copy number variation loci, or in heterogeneous tumor sequences.

Genome sequence of Comamonas testosteroni ATCC 11996, a representative strain involved in steroid degradation.

Comamonas testosteroni strains belong to the family of Comamonadaceae and are known for their ability to utilize steroid compounds as carbon source. Here, we present the draft genome sequence of strain ATCC 11996, with a G+C content of 61.48%.

Accurate variant detection across non-amplified and whole genome amplified DNA using targeted next generation sequencing.

BACKGROUND: Many hypothesis-driven genetic studies require the ability to comprehensively and efficiently target specific regions of the genome to detect sequence variations. Often, sample availability is limited requiring the use of whole genome amplification (WGA). We evaluated a high-throughput microdroplet-based PCR approach in combination with next generation sequencing (NGS) to target 384 discrete exons from 373 genes involved in cancer. In our evaluation, we compared the performance of six non-amplified gDNA samples from two HapMap family trios. Three of these samples were also preamplified by WGA and evaluated. We tested sample pooling or multiplexing strategies at different stages of the tested targeted NGS (T-NGS) workflow. RESULTS: The results demonstrated comparable sequence performance between non-amplified and preamplified samples and between different indexing strategies [sequence specificity of 66.0% ± 3.4%, uniformity (coverage at 0.2× of the mean) of 85.6% ± 0.6%]. The average genotype concordance maintained across all the samples was 99.5% ± 0.4%, regardless of sample type or pooling strategy. We did not detect any errors in the Mendelian patterns of inheritance of genotypes between the parents and offspring within each trio. We also demonstrated the ability to detect minor allele frequencies within the pooled samples that conform to predicted models. CONCLUSION: Our described PCR-based sample multiplex approach and the ability to use WGA material for NGS may enable researchers to perform deep resequencing studies and explore variants at very low frequencies and cost.

A tissue-specific landscape of sense/antisense transcription in the mouse intestine.

BACKGROUND: The intestinal mucosa is characterized by complex metabolic and immunological processes driven highly dynamic gene expression programs. With the advent of next generation sequencing and its utilization for the analysis of the RNA sequence space, the level of detail on the global architecture of the transcriptome reached a new order of magnitude compared to microarrays. RESULTS: We report the ultra-deep characterization of the polyadenylated transcriptome in two closely related, yet distinct regions of the mouse intestinal tract (small intestine and colon). We assessed tissue-specific transcriptomal architecture and the presence of novel transcriptionally active regions (nTARs). In the first step, signatures of 20,541 NCBI RefSeq transcripts could be identified in the intestine (74.1% of annotated genes), thereof 16,742 are common in both tissues. Although the majority of reads could be linked to annotated genes, 27,543 nTARs not consistent with current gene annotations in RefSeq or ENSEMBL were identified. By use of a second independent strand-specific RNA-Seq protocol, 20,966 of these nTARs were confirmed, most of them in vicinity of known genes. We further categorized our findings by their relative adjacency to described exonic elements and investigated regional differences of novel transcribed elements in small intestine and colon. CONCLUSIONS: The current study demonstrates the complexity of an archetypal mammalian intestinal mRNA transcriptome in high resolution and identifies novel transcriptionally active regions at strand-specific, single base resolution. Our analysis for the first time shows a strand-specific comparative picture of nTARs in two tissues and represents a resource for further investigating the transcriptional processes that contribute to tissue identity.

SNP discovery performance of two second-generation sequencing platforms in the NOD2 gene region.

A potentially important application of second generation sequencing technologies is to identify disease-associated variation. For comparison of the performance in SNP detection, the Crohn's disease (CD)-associated NOD2 gene was subjected to targeted resequencing using two different second-generation sequencing technologies. Eleven CD patients were selected based on their haplotype background at the NOD2 locus. The 40-kb large NOD2 gene region was amplified using long-range PCR (LR-PCR), and sequenced with the Roche 454/FLX system, an Applied Biosystems SOLiD mate-pair library (2 x 25 bp), and a SOLiD fragment (50 bp) library. The entire NOD2 region was also sequenced using conventional Sanger technology. Four-hundred forty-two single nucleotide polymorphisms (SNPs) were discovered with the SOLiD mate-pair library, 454 with the fragment library, and 441 with the 454/FLX. For the homozygous SNPs, 98% were confirmed by Sanger for the mate-pair library, 100% for the fragment library and 99% for the 454/FLX. Ninety-six percent of the heterozygous SNPs detected with the SOLiD mate-pair library, 91% with the fragment library and 96% with the 454/FLX were confirmed by Sanger. In a simulation, the SNP detection performance fell rapidly when the achieved coverage was below 40 x. Due to uneven representation of the target region when using LR-PCR, oversequencing of other regions is necessary.

Genomic and transcriptomic changes complement each other in the pathogenesis of sporadic Burkitt lymphoma.

Burkitt lymphoma (BL) is the most common B-cell lymphoma in children. Within the International Cancer Genome Consortium (ICGC), we performed whole genome and transcriptome sequencing of 39 sporadic BL. Here, we unravel interaction of structural, mutational, and transcriptional changes, which contribute to MYC oncogene dysregulation together with the pathognomonic IG-MYC translocation. Moreover, by mapping IGH translocation breakpoints, we provide evidence that the precursor of at least a subset of BL is a B-cell poised to express IGHA. We describe the landscape of mutations, structural variants, and mutational processes, and identified a series of driver genes in the pathogenesis of BL, which can be targeted by various mechanisms, including IG-non MYC translocations, germline and somatic mutations, fusion transcripts, and alternative splicing.

De novo assembly and annotation of the European abalone Haliotis tuberculata transcriptome.

The European abalone Haliotis tuberculata is a delicacy and consequently a commercially valuable gastropod species. Aquaculture production and wild populations are subjected to multiple climate-associated stressors and anthropogenic pressures, including rising sea-surface temperatures, ocean acidification and an emerging pathogenic Vibrio infection. Transcript expression data provides a valuable resource for understanding abalone responses to variation in the biotic and abiotic environment. To generate an extensive transcriptome, we performed next-generation sequencing of RNA on larvae exposed to temperature and pH variation and on haemolymph of adults from two wild populations after experimental infection with Vibrio harveyi. We obtained more than 1.5 billion raw paired-end reads, which were assembled into 328,519 contigs. Filtration and clustering produced a transcriptome of 41,099 transcripts, of which 10,626 (25.85%) were annotated with Blast hits, and 7380 of these were annotated with Gene Ontology (GO) terms in Blast2Go. A differential expression analysis comparing all samples from the two life stages identified 5690 and 10,759 transcripts with significantly higher expression in larvae and adult haemolymph respectively. This is the greatest sequencing effort yet in the Haliotis genus, and provides the first high-throughput transcriptomic resource for H. tuberculata.

Biophysical and Population Genetic Models Predict the Presence of "Phantom" Stepping Stones Connecting Mid-Atlantic Ridge Vent Ecosystems.

Deep-sea hydrothermal vents are patchily distributed ecosystems inhabited by specialized animal populations that are textbook meta-populations. Many vent-associated species have free-swimming, dispersive larvae that can establish connections between remote populations. However, connectivity patterns among hydrothermal vents are still poorly understood because the deep sea is undersampled, the molecular tools used to date are of limited resolution, and larval dispersal is difficult to measure directly. A better knowledge of connectivity is urgently needed to develop sound environmental management plans for deep-sea mining. Here, we investigated larval dispersal and contemporary connectivity of ecologically important vent mussels (Bathymodiolus spp.) from the Mid-Atlantic Ridge by using high-resolution ocean modeling and population genetic methods. Even when assuming a long pelagic larval duration, our physical model of larval drift suggested that arrival at localities more than 150 km from the source site is unlikely and that dispersal between populations requires intermediate habitats ("phantom" stepping stones). Dispersal patterns showed strong spatiotemporal variability, making predictions of population connectivity challenging. The assumption that mussel populations are only connected via additional stepping stones was supported by contemporary migration rates based on neutral genetic markers. Analyses of population structure confirmed the presence of two southern and two hybridizing northern mussel lineages that exhibited a substantial, though incomplete, genetic differentiation. Our study provides insights into how vent animals can disperse between widely separated vent habitats and shows that recolonization of perturbed vent sites will be subject to chance events, unless connectivity is explicitly considered in the selection of conservation areas.

Influence of a non-hospital medical care facility on antimicrobial resistance in wastewater.

The global widespread use of antimicrobials and accompanying increase in resistant bacterial strains is of major public health concern. Wastewater systems and wastewater treatment plants are considered a niche for antibiotic resistance genes (ARGs), with diverse microbial communities facilitating ARG transfer via mobile genetic element (MGE). In contrast to hospital sewage, wastewater from other health care facilities is still poorly investigated. At the instance of a nursing home located in south-west Germany, in the present study, shotgun metagenomics was used to investigate the impact on wastewater of samples collected up- and down-stream in different seasons. Microbial composition, ARGs and MGEs were analyzed using different annotation approaches with various databases, including Antibiotic Resistance Ontologies (ARO), integrons and plasmids. Our analysis identified seasonal differences in microbial communities and abundance of ARG and MGE between samples from different seasons. However, no obvious differences were detected between up- and downstream samples. The results suggest that, in contrast to hospitals, sewage from the nursing home does not have a major impact on ARG or MGE in wastewater, presumably due to much less intense antimicrobial usage. Possible limitations of metagenomic studies using high-throughput sequencing for detection of genes that seemingly confer antibiotic resistance are discussed.

The Magellania venosa Biomineralizing Proteome: A Window into Brachiopod Shell Evolution.

Brachiopods are a lineage of invertebrates well known for the breadth and depth of their fossil record. Although the quality of this fossil record attracts the attention of paleontologists, geochemists, and paleoclimatologists, modern day brachiopods are also of interest to evolutionary biologists due to their potential to address a variety of questions ranging from developmental biology to biomineralization. The brachiopod shell is a composite material primarily composed of either calcite or calcium phosphate in close association with proteins and polysaccharides which give these composite structures their material properties. The information content of these biomolecules, sequestered within the shell during its construction, has the potential to inform hypotheses focused on describing how brachiopod shell formation evolved. Here, using high throughput proteomic approaches and next generation sequencing, we have surveyed and characterized the first shell-proteome and shell-forming transcriptome of any brachiopod, the South American Magellania venosa (Rhynchonelliformea: Terebratulida). We find that the seven most abundant proteins present in the shell are unique to M. venosa, but that these proteins display biochemical features found in other metazoan biomineralization proteins. We can also detect some M. venosa proteins that display significant sequence similarity to other metazoan biomineralization proteins, suggesting that some elements of the brachiopod shell-forming proteome are deeply evolutionarily conserved. We also employed a variety of preparation methods to isolate shell proteins and find that in comparison to the shells of other spiralian invertebrates (such as mollusks) the shell ultrastructure of M. venosa may explain the effects these preparation strategies have on our results.

Abundant toxin-related genes in the genomes of beneficial symbionts from deep-sea hydrothermal vent mussels.

Bathymodiolus mussels live in symbiosis with intracellular sulfur-oxidizing (SOX) bacteria that provide them with nutrition. We sequenced the SOX symbiont genomes from two Bathymodiolus species. Comparison of these symbiont genomes with those of their closest relatives revealed that the symbionts have undergone genome rearrangements, and up to 35% of their genes may have been acquired by horizontal gene transfer. Many of the genes specific to the symbionts were homologs of virulence genes. We discovered an abundant and diverse array of genes similar to insecticidal toxins of nematode and aphid symbionts, and toxins of pathogens such as Yersinia and Vibrio. Transcriptomics and proteomics revealed that the SOX symbionts express the toxin-related genes (TRGs) in their hosts. We hypothesize that the symbionts use these TRGs in beneficial interactions with their host, including protection against parasites. This would explain why a mutualistic symbiont would contain such a remarkable 'arsenal' of TRGs.

Effect of sludge age on population dynamics and acetate utilization kinetics under aerobic conditions.

The study addressed acetate utilization by an acclimated mixed microbial culture under different growth conditions. It explored changes in the composition of the microbial community and variable process kinetics induced by different culture history. Sequencing batch reactors were operated at steady-state at different sludge ages of two and ten days. Microbial population structure was determined using high-throughput sequencing of 16S rRNA genes. Parallel batch experiments were conducted with acclimated biomass for respirometric analyses. A lower sludge age sustained a different community, which also reflected as variable kinetics for microbial growth and biopolymer storage. The maximum growth rate was observed to change from 3.9/d to 8.5/d and the substrate storage rate from 3.5/d to 5.9/d when the sludge age was decreased from 10 d to 2.0 d. Results challenge the basic definition of heterotrophic biomass in activated sludge models, at least by means of variable kinetics under different growth conditions.

Giant hydrogen sulfide plume in the oxygen minimum zone off Peru supports chemolithoautotrophy.

In Eastern Boundary Upwelling Systems nutrient-rich waters are transported to the ocean surface, fuelling high photoautotrophic primary production. Subsequent heterotrophic decomposition of the produced biomass increases the oxygen-depletion at intermediate water depths, which can result in the formation of oxygen minimum zones (OMZ). OMZs can sporadically accumulate hydrogen sulfide (H2S), which is toxic to most multicellular organisms and has been implicated in massive fish kills. During a cruise to the OMZ off Peru in January 2009 we found a sulfidic plume in continental shelf waters, covering an area >5500 km(2), which contained ∼2.2×10(4) tons of H2S. This was the first time that H2S was measured in the Peruvian OMZ and with ∼440 km(3) the largest plume ever reported for oceanic waters. We assessed the phylogenetic and functional diversity of the inhabiting microbial community by high-throughput sequencing of DNA and RNA, while its metabolic activity was determined with rate measurements of carbon fixation and nitrogen transformation processes. The waters were dominated by several distinct γ-, δ- and ε-proteobacterial taxa associated with either sulfur oxidation or sulfate reduction. Our results suggest that these chemolithoautotrophic bacteria utilized several oxidants (oxygen, nitrate, nitrite, nitric oxide and nitrous oxide) to detoxify the sulfidic waters well below the oxic surface. The chemolithoautotrophic activity at our sampling site led to high rates of dark carbon fixation. Assuming that these chemolithoautotrophic rates were maintained throughout the sulfidic waters, they could be representing as much as ∼30% of the photoautotrophic carbon fixation. Postulated changes such as eutrophication and global warming, which lead to an expansion and intensification of OMZs, might also increase the frequency of sulfidic waters. We suggest that the chemolithoautotrophically fixed carbon may be involved in a negative feedback loop that could fuel further sulfate reduction and potentially stabilize the sulfidic OMZ waters.

Small changes in pH have direct effects on marine bacterial community composition: a microcosm approach.

As the atmospheric CO(2) concentration rises, more CO(2) will dissolve in the oceans, leading to a reduction in pH. Effects of ocean acidification on bacterial communities have mainly been studied in biologically complex systems, in which indirect effects, mediated through food web interactions, come into play. These approaches come close to nature but suffer from low replication and neglect seasonality. To comprehensively investigate direct pH effects, we conducted highly-replicated laboratory acidification experiments with the natural bacterial community from Helgoland Roads (North Sea). Seasonal variability was accounted for by repeating the experiment four times (spring, summer, autumn, winter). Three dilution approaches were used to select for different ecological strategies, i.e. fast-growing or low-nutrient adapted bacteria. The pH levels investigated were in situ seawater pH (8.15-8.22), pH 7.82 and pH 7.67, representing the present-day situation and two acidification scenarios projected for the North Sea for the year 2100. In all seasons, both automated ribosomal intergenic spacer analysis and 16S ribosomal amplicon pyrosequencing revealed pH-dependent community shifts for two of the dilution approaches. Bacteria susceptible to changes in pH were different members of Gammaproteobacteria, Flavobacteriaceae, Rhodobacteraceae, Campylobacteraceae and further less abundant groups. Their specific response to reduced pH was often context-dependent. Bacterial abundance was not influenced by pH. Our findings suggest that already moderate changes in pH have the potential to cause compositional shifts, depending on the community assembly and environmental factors. By identifying pH-susceptible groups, this study provides insights for more directed, in-depth community analyses in large-scale and long-term experiments.

Genome and low-iron response of an oceanic diatom adapted to chronic iron limitation.

BACKGROUND: Biogeochemical elemental cycling is driven by primary production of biomass via phototrophic phytoplankton growth, with 40% of marine productivity being assigned to diatoms. Phytoplankton growth is widely limited by the availability of iron, an essential component of the photosynthetic apparatus. The oceanic diatom Thalassiosira oceanica shows a remarkable tolerance to low-iron conditions and was chosen as a model for deciphering the cellular response upon shortage of this essential micronutrient. RESULTS: The combined efforts in genomics, transcriptomics and proteomics reveal an unexpected metabolic flexibility in response to iron availability for T. oceanica CCMP1005. The complex response comprises cellular retrenchment as well as remodeling of bioenergetic pathways, where the abundance of iron-rich photosynthetic proteins is lowered, whereas iron-rich mitochondrial proteins are preserved. As a consequence of iron deprivation, the photosynthetic machinery undergoes a remodeling to adjust the light energy utilization with the overall decrease in photosynthetic electron transfer complexes. CONCLUSIONS: Beneficial adaptations to low-iron environments include strategies to lower the cellular iron requirements and to enhance iron uptake. A novel contribution enhancing iron economy of phototrophic growth is observed with the iron-regulated substitution of three metal-containing fructose-bisphosphate aldolases involved in metabolic conversion of carbohydrates for enzymes that do not contain metals. Further, our data identify candidate components of a high-affinity iron-uptake system, with several of the involved genes and domains originating from duplication events. A high genomic plasticity, as seen from the fraction of genes acquired through horizontal gene transfer, provides the platform for these complex adaptations to a low-iron world.

High tandem repeat content in the genome of the short-lived annual fish Nothobranchius furzeri: a new vertebrate model for aging research.

BACKGROUND: The annual fish Nothobranchius furzeri is the vertebrate with the shortest known life span in captivity. Fish of the GRZ strain live only three to four months under optimal laboratory conditions, show explosive growth, early sexual maturation and age-dependent physiological and behavioral decline, and express aging related biomarkers. Treatment with resveratrol and low temperature significantly extends the maximum life span. These features make N. furzeri a promising new vertebrate model for age research. RESULTS: To contribute to establishing N. furzeri as a new model organism, we provide a first insight into its genome and a comparison to medaka, stickleback, tetraodon and zebrafish. The N. furzeri genome contains 19 chromosomes (2n = 38). Its genome of between 1.6 and 1.9 Gb is the largest among the analyzed fish species and has, at 45%, the highest repeat content. Remarkably, tandem repeats comprise 21%, which is 4-12 times more than in the other four fish species. In addition, G+C-rich tandem repeats preferentially localize to centromeric regions. Phylogenetic analysis based on coding sequences identifies medaka as the closest relative. Genotyping of an initial set of 27 markers and multi-locus fingerprinting of one microsatellite provides the first molecular evidence that the GRZ strain is highly inbred. CONCLUSIONS: Our work presents a first basis for systematic genomic and genetic analyses aimed at understanding the mechanisms of life span determination in N. furzeri.