Sequence verification is essential for plasmids used as critical reagents or therapeutic products. Typically, high-quality plasmid sequence is achieved through capillary-based Sanger sequencing, ...requiring customized sets of primers for each plasmid. This process can become expensive, particularly for applications where the validated sequence needs to be produced within a regulated and quality-controlled environment for downstream clinical research applications.
Here, we describe a cost-effective and accurate plasmid sequencing and consensus generation procedure using the Oxford Nanopore Technologies' MinION device as an alternative to capillary-based plasmid sequencing options. This procedure can verify the identity of a pure population of plasmid, either confirming it matches the known and expected sequence, or identifying mutations present in the plasmid if any exist. We use a full MinION flow cell per plasmid, maximizing available data and allowing for stringent quality filters. Pseudopairing reads for consensus base calling reduces read error rates from 5.3 to 0.53%, and our pileup consensus approach provides per-base counts and confidence scores, allowing for interpretation of the certainty of the resulting consensus sequences. For pure plasmid samples, we demonstrate 100% accuracy in the resulting consensus sequence, and the sensitivity to detect small mutations such as insertions, deletions, and single nucleotide variants. In test cases where the sequenced pool of plasmids contains subclonal templates, detection sensitivity is similar to that of traditional capillary sequencing.
Our pipeline can provide significant cost savings compared to outsourcing clinical-grade sequencing of plasmids, making generation of high-quality plasmid sequence for clinical sequence verification more accessible. While other long-read-based methods offer higher-throughput and less cost, our pipeline produces complete and accurate sequence verification for cases where absolute sequence accuracy is required.
Multiple hypotheses have been proposed to explain the annual migration of the Serengeti wildebeest, but few studies have compared distribution patterns with environmental drivers. We used a ...rainfall‐driven model of grass dynamics and wildebeest movement to generate simulated monthly wildebeest distributions, with wildebeest movement decisions depending on 14 candidate models of adaptive movement in response to resource availability. We used information‐theoretic approaches to compare the fits of simulated and observed monthly distribution patterns at two spatial scales over a 3‐year period. Models that included the intake rate and nitrogen (N) concentration of green grass and the suppressive effect of tree cover on grass biomass provided the best model fits at both spatial scales tested, suggesting that digestive constraints and protein requirements may play key roles in driving migratory behavior. The emergence of a migration was predicted to be dependent on the ability of the wildebeest to track changes in resource abundance at relatively large scales (>80–100 km). When movement decisions are based solely on local resource availability, the wildebeest fail to migrate across the ecosystem. Our study highlights the potentially key role of strong and countervailing seasonally driven rainfall and fertility gradients—a consistent feature of African savanna ecosystems—as drivers of long‐distance seasonal migrations in ungulates.
The diversity of life is ultimately generated by evolution, and much attention has focused on the rapid evolution of ecological traits. Yet, the tendency for many ecological traits to instead remain ...similar over time niche conservatism (NC) has many consequences for the fundamental patterns and processes studied in ecology and conservation biology. Here, we describe the mounting evidence for the importance of NC to major topics in ecology (e.g. species richness, ecosystem function) and conservation (e.g. climate change, invasive species). We also review other areas where it may be important but has generally been overlooked, in both ecology (e.g. food webs, disease ecology, mutualistic interactions) and conservation (e.g. habitat modification). We summarize methods for testing for NC, and suggest that a commonly used and advocated method (involving a test for phylogenetic signal) is potentially problematic, and describe alternative approaches. We suggest that considering NC: (1) focuses attention on the within-species processes that cause traits to be conserved over time, (2) emphasizes connections between questions and research areas that are not obviously related (e.g. invasives, global warming, tropical richness), and (3) suggests new areas for research (e.g. why are some clades largely nocturnal? why do related species share diseases?).
Somatic missense mutations can initiate tumorogenesis and, conversely, anti-tumor cytotoxic T cell (CTL) responses. Tumor genome analysis has revealed extreme heterogeneity among tumor missense ...mutation profiles, but their relevance to tumor immunology and patient outcomes has awaited comprehensive evaluation. Here, for 515 patients from six tumor sites, we used RNA-seq data from The Cancer Genome Atlas to identify mutations that are predicted to be immunogenic in that they yielded mutational epitopes presented by the MHC proteins encoded by each patient's autologous HLA-A alleles. Mutational epitopes were associated with increased patient survival. Moreover, the corresponding tumors had higher CTL content, inferred from CD8A gene expression, and elevated expression of the CTL exhaustion markers PDCD1 and CTLA4. Mutational epitopes were very scarce in tumors without evidence of CTL infiltration. These findings suggest that the abundance of predicted immunogenic mutations may be useful for identifying patients likely to benefit from checkpoint blockade and related immunotherapies.
Interactions between natural enemies and their victims are a pervasive feature of the natural world. In this paper, we discuss trophic interactions as determinants of geographic range limits. ...Predators can directly limit ranges, or do so in conjunction with competition. Dispersal can at times permit a specialist predator to constrain the distribution of its prey-and thus itself-along a gradient. Conversely, we suggest that predators can also at times permit prey to have larger ranges than would be seen without predation. We discuss several ecological and evolutionary mechanisms that can lead to this counter-intuitive outcome.
Invasive lobular carcinoma (ILC) is the second most prevalent histologic subtype of invasive breast cancer. Here, we comprehensively profiled 817 breast tumors, including 127 ILC, 490 ductal (IDC), ...and 88 mixed IDC/ILC. Besides E-cadherin loss, the best known ILC genetic hallmark, we identified mutations targeting PTEN, TBX3, and FOXA1 as ILC enriched features. PTEN loss associated with increased AKT phosphorylation, which was highest in ILC among all breast cancer subtypes. Spatially clustered FOXA1 mutations correlated with increased FOXA1 expression and activity. Conversely, GATA3 mutations and high expression characterized luminal A IDC, suggesting differential modulation of ER activity in ILC and IDC. Proliferation and immune-related signatures determined three ILC transcriptional subtypes associated with survival differences. Mixed IDC/ILC cases were molecularly classified as ILC-like and IDC-like revealing no true hybrid features. This multidimensional molecular atlas sheds new light on the genetic bases of ILC and provides potential clinical options.
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•Invasive lobular carcinoma (ILC) is a clinically and molecularly distinct disease•ILCs show CDH1 and PTEN loss, AKT activation, and mutations in TBX3 and FOXA1•Proliferation and immune-related gene expression signatures define 3 ILC subtypes•Genetic features classify mixed tumors into lobular-like and ductal-like subgroups
A comprehensive analysis of 817 breast tumor samples determines invasive lobular carcinoma as a molecularly distinct disease with characteristic genetic features, providing key information for patient stratification that may allow a more informed clinical follow-up.
Opportunities to conduct large-scale field experiments are rare, but provide a unique opportunity to reveal the complex processes that operate within natural ecosystems. Here, we review the design of ...existing, large-scale forest fragmentation experiments. Based on this review, we develop a design for the Stability of Altered Forest Ecosystems (SAFE) Project, a new forest fragmentation experiment to be located in the lowland tropical forests of Borneo (Sabah, Malaysia). The SAFE Project represents an advance on existing experiments in that it: (i) allows discrimination of the effects of landscape-level forest cover from patch-level processes; (ii) is designed to facilitate the unification of a wide range of data types on ecological patterns and processes that operate over a wide range of spatial scales; (iii) has greater replication than existing experiments; (iv) incorporates an experimental manipulation of riparian corridors; and (v) embeds the experimentally fragmented landscape within a wider gradient of land-use intensity than do existing projects. The SAFE Project represents an opportunity for ecologists across disciplines to participate in a large initiative designed to generate a broad understanding of the ecological impacts of tropical forest modification.
Habitat construction and phenotypic plasticity are alternative responses to variable environments. We explored evolution along an environmental gradient of habitat construction alone and in ...combination with phenotypic plasticity using individual-based simulations that manipulated the fitness benefit of construction and whether construction maintained or eliminated that gradient. Construction was favored when its benefits were more likely to flow to the immediate offspring of the constructing individuals. Habitat construction and phenotypic plasticity traded off against each other or plasticity was selected against, depending on how the optimum environment varied and with the fitness value of construction. When selection favored differences in the amount of construction along the environmental gradient, genetic differentiation for habitat construction increased as the fitness value of construction increased. The degree to which each adaptive response was likely to evolve also depended on the precise ordering of life history events. Adaptive habitat construction does not always occur and may be selected against.
Recent controversy over whether biodiversity reduces disease risk (dilution effect) has focused on the ecology of Lyme disease, a tick-borne zoonosis. A criticism of the dilution effect is that ...increasing host species richness might amplify disease risk, assuming that total host abundance, and therefore feeding opportunities for ticks, increase with species richness. In contrast, a dilution effect is expected when poor quality hosts for ticks and pathogens (dilution hosts) divert tick blood meals away from competent hosts. Even if host densities are additive, the relationship between host density and tick encounters can be nonlinear if the number of ticks that encounter a host is a saturating function of host density, which occurs if ticks aggregate on the remaining hosts rather than failing to find a host before death. Both dilution and amplification are theoretical possibilities, and assessing which is more prevalent required detailed analyses of empirical systems. We used field data to explore the degree of tick redistribution onto fewer individuals with variation in intraspecific host density and novel data-driven models for tick dynamics to determine how changes in vertebrate community composition influence the density of nymphs infected with the Lyme bacterium. To be conservative, we allowed total host density to increase additively with species richness. Our long-term field studies found that larval and nymphal ticks redistribute onto fewer individuals as host densities decline, that a large proportion of nymphs and adults find hosts, and that mice and chipmunks feed a large proportion of nymphs. White-footed mice, eastern chipmunks, short-tailed shrews, and masked shrews were important amplification hosts that greatly increased the density of infected nymphs. Gray squirrels and Virginia opossums were important dilution hosts. Removing these two species increased the maximum number of larvae attached to amplification hosts by 57%. Raccoons and birds were minor dilution hosts under some conditions. Even under the assumption of additive community assembly, some species are likely to reduce the density of infected nymphs as diversity increases. If the assumption of additivity is relaxed, then species that reduce the density of small mammals through predation or competition might substantially reduce disease risk.
Phenotypic plasticity in evolutionary rescue experiments Chevin, Luis-Miguel; Gallet, Romain; Gomulkiewicz, Richard ...
Philosophical transactions - Royal Society. Biological sciences,
01/2013, Letnik:
368, Številka:
1610
Journal Article
Recenzirano
Odprti dostop
Population persistence in a new and stressful environment can be influenced by the plastic phenotypic responses of individuals to this environment, and by the genetic evolution of plasticity itself. ...This process has recently been investigated theoretically, but testing the quantitative predictions in the wild is challenging because (i) there are usually not enough population replicates to deal with the stochasticity of the evolutionary process, (ii) environmental conditions are not controlled, and (iii) measuring selection and the inheritance of traits affecting fitness is difficult in natural populations. As an alternative, predictions from theory can be tested in the laboratory with controlled experiments. To illustrate the feasibility of this approach, we briefly review the literature on the experimental evolution of plasticity, and on evolutionary rescue in the laboratory, paying particular attention to differences and similarities between microbes and multicellular eukaryotes. We then highlight a set of questions that could be addressed using this framework, which would enable testing the robustness of theoretical predictions, and provide new insights into areas that have received little theoretical attention to date.
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