Oxford Nanopore's third-generation single-molecule sequencing platform promises to decrease costs for reagents and instrumentation. After a 2-year hiatus following the initial announcement, the first devices have been released as part of an early access program. We explore the performance of this platform by resequencing the lambda phage genome, and amplicons from a snake venom gland transcriptome. Although the handheld MinION sequencer can generate more than 150 megabases of raw data in one run, at most a quarter of the resulting reads map to the reference, with less than average 10% identity. Much of the sequence consists of insertion/deletion errors, or is seemingly without similarity to the template. Using the lambda phage data as an example, although the reads are long, averaging 5 kb, at best 890 ± 1932 bases per mapped read could be matched to the reference without soft clipping. In the course of a 36 h run on the MinION, it was possible to resequence the 48 kb lambda phage reference at 16× coverage. Currently, substantially larger projects would not be feasible using the MinION. Without increases in accuracy, which would be required for applications such as genome scaffolding and phasing, the current utility of the MinION appears limited. Library preparation requires access to a molecular laboratory, and is of similar complexity and cost to that of other next-generation sequencing platforms. The MinION is an exciting step in a new direction for single-molecule sequencing, though it will require dramatic decreases in error rates before it lives up to its promise.

Ancient and archival DNA samples are valuable resources for the study of diverse historical processes. In particular, museum specimens provide access to biotas distant in time and space, and can provide insights into ecological and evolutionary changes over time. However, archival specimens are difficult to handle; they are often fragile and irreplaceable, and typically contain only short segments of denatured DNA. Here we present a set of tools for processing such samples for state-of-the-art genetic analysis. First, we report a protocol for minimally destructive DNA extraction of insect museum specimens, which produced sequenceable DNA from all of the samples assayed. The 11 specimens analyzed had fragmented DNA, rarely exceeding 100 bp in length, and could not be amplified by conventional PCR targeting the mitochondrial cytochrome oxidase I gene. Our approach made these samples amenable to analysis with commonly used next-generation sequencing-based molecular analytic tools, including RAD-tagging and shotgun genome re-sequencing. First, we used museum ant specimens from three species, each with its own reference genome, for RAD-tag mapping. Were able to use the degraded DNA sequences, which were sequenced in full, to identify duplicate reads and filter them prior to base calling. Second, we re-sequenced six Hawaiian Drosophila species, with millions of years of divergence, but with only a single available reference genome. Despite a shallow coverage of 0.37±0.42 per base, we could recover a sufficient number of overlapping SNPs to fully resolve the species tree, which was consistent with earlier karyotypic studies, and previous molecular studies, at least in the regions of the tree that these studies could resolve. Although developed for use with degraded DNA, all of these techniques are readily applicable to more recent tissue, and are suitable for liquid handling automation.

BackgroundReproductive division of labor in eusocial insects is a striking example of a shared genetic background giving rise to alternative phenotypes, namely queen and worker castes. Queen and worker phenotypes play major roles in the evolution of eusocial insects. Their behavior, morphology and physiology underpin many ecologically relevant colony-level traits, which evolved in parallel in multiple species.ResultsUsing queen and worker transcriptomic data from 16 ant species we tested the hypothesis that conserved sets of genes are involved in ant reproductive division of labor. We further hypothesized that such sets of genes should also be involved in the parallel evolution of other key traits. We applied weighted gene co-expression network analysis, which clusters co-expressed genes into modules, whose expression levels can be summarized by their ‘eigengenes’. Eigengenes of most modules were correlated with phenotypic differentiation between queens and workers. Furthermore, eigengenes of some modules were correlated with repeated evolution of key phenotypes such as complete worker sterility, the number of queens per colony, and even invasiveness. Finally, connectivity and expression levels of genes within the co-expressed network were strongly associated with the strength of selection. Although caste-associated sets of genes evolve faster than non-caste-associated, we found no evidence for queen- or worker-associated co-expressed genes evolving faster than one another.ConclusionsThese results identify conserved functionally important genomic units that likely serve as building blocks of phenotypic innovation, and allow the remarkable breadth of parallel evolution seen in ants, and possibly other eusocial insects as well.Electronic supplementary materialThe online version of this article (doi:10.1186/s13059-016-0902-7) contains supplementary material, which is available to authorized users.

RAD-tag is a powerful tool for high-throughput genotyping. It relies on PCR amplification of the starting material, following enzymatic digestion and sequencing adaptor ligation. Amplification introduces duplicate reads into the data, which arise from the same template molecule and are statistically nonindependent, potentially introducing errors into genotype calling. In shotgun sequencing, data duplicates are removed by filtering reads starting at the same position in the alignment. However, restriction enzymes target specific locations within the genome, causing reads to start in the same place, and making it difficult to estimate the extent of PCR duplication. Here, we introduce a slight change to the Illumina sequencing adaptor chemistry, appending a unique four-base tag to the first index read, which allows duplicate discrimination in aligned data. This approach was validated on the Illumina MiSeq platform, using double-digest libraries of ants (Wasmannia auropunctata) and yeast (Saccharomyces cerevisiae) with known genotypes, producing modest though statistically significant gains in the odds of calling a genotype accurately. More importantly, removing duplicates also corrected for strong sample-to-sample variability of genotype calling accuracy seen in the ant samples. For libraries prepared from low-input degraded museum bird samples (Mixornis gularis), which had low complexity, having been generated from relatively few starting molecules, adaptor tags show that virtually all of the genotypes were called with inflated confidence as a result of PCR duplicates. Quantification of library complexity by adaptor tagging does not significantly increase the difficulty of the overall workflow or its cost, but corrects for differences in quality between samples and permits analysis of low-input material.

BackgroundWhile many studies have shown that extracellular proteins evolve rapidly, how selection acts on them remains poorly understood. We used snake venoms to understand the interaction between ecology, expression level, and evolutionary rate in secreted protein systems. Venomous snakes employ well-integrated systems of proteins and organic constituents to immobilize prey. Venoms are generally optimized to subdue preferred prey more effectively than non-prey, and many venom protein families manifest positive selection and rapid gene family diversification. Although previous studies have illuminated how individual venom protein families evolve, how selection acts on venoms as integrated systems, is unknown.ResultsUsing next-generation transcriptome sequencing and mass spectrometry, we examined microevolution in two pitvipers, allopatrically separated for at least 1.6 million years, and their hybrids. Transcriptomes of parental species had generally similar compositions in regard to protein families, but for a given protein family, the homologs present and concentrations thereof sometimes differed dramatically. For instance, a phospholipase A2 transcript comprising 73.4 % of the Protobothrops elegans transcriptome, was barely present in the P. flavoviridis transcriptome (<0.05 %). Hybrids produced most proteins found in both parental venoms. Protein evolutionary rates were positively correlated with transcriptomic and proteomic abundances, and the most abundant proteins showed positive selection. This pattern holds with the addition of four other published crotaline transcriptomes, from two more genera, and also for the recently published king cobra genome, suggesting that rapid evolution of abundant proteins may be generally true for snake venoms. Looking more broadly at Protobothrops, we show that rapid evolution of the most abundant components is due to positive selection, suggesting an interplay between abundance and adaptation.ConclusionsGiven log-scale differences in toxin abundance, which are likely correlated with biosynthetic costs, we hypothesize that as a result of natural selection, snakes optimize return on energetic investment by producing more of venom proteins that increase their fitness. Natural selection then acts on the additive genetic variance of these components, in proportion to their contributions to overall fitness. Adaptive evolution of venoms may occur most rapidly through changes in expression levels that alter fitness contributions, and thus the strength of selection acting on specific secretome components.Electronic supplementary materialThe online version of this article (doi:10.1186/s12864-015-1832-6) contains supplementary material, which is available to authorized users.

Understanding genetic changes caused by novel pathogens and parasites can reveal mechanisms of adaptation and genetic robustness. Using whole-genome sequencing of museum and modern specimens, we describe the genomic changes in a wild population of honey bees in North America following the introduction of the ectoparasitic mite, Varroa destructor. Even though colony density in the study population is the same today as in the past, a major loss of haplotypic diversity occurred, indicative of a drastic mitochondrial bottleneck, caused by massive colony mortality. In contrast, nuclear genetic diversity did not change, though hundreds of genes show signs of selection. The genetic diversity within each bee colony, particularly as a consequence of polyandry by queens, may enable preservation of genetic diversity even during population bottlenecks. These findings suggest that genetically diverse honey bee populations can recover from introduced diseases by evolving rapid tolerance, while maintaining much of the standing genetic variation.

The sophisticated colony organization of eusocial insects is primarily maintained through the utilization of pheromones. The regulation of these complex social interactions requires intricate chemoreception systems. The recent publication of the genome of Zootermopsis nevadensis opened a new avenue to study molecular basis of termite caste systems. Although there has been a growing interest in the termite chemoreception system that regulates their sophisticated caste system, the relationship between division of labor and expression of chemoreceptor genes remains to be explored. Using high-throughput mRNA sequencing (RNA-seq), we found several chemoreceptors that are differentially expressed among castes and between sexes in a subterranean termite Reticulitermes speratus. In total, 53 chemoreception-related genes were annotated, including 22 odorant receptors, 7 gustatory receptors, 12 ionotropic receptors, 9 odorant-binding proteins, and 3 chemosensory proteins. Most of the chemoreception-related genes had caste-related and sex-related expression patterns; in particular, some chemoreception genes showed king-biased or queen-biased expression patterns. Moreover, more than half of the genes showed significant age-dependent differences in their expression in female and/or male reproductives. These results reveal a strong relationship between the evolution of the division of labor and the regulation of chemoreceptor gene expression, thereby demonstrating the chemical communication and underlining chemoreception mechanism in social insects.

Queen-worker differentiation in eusocial organisms may have originated from decoupling of maternal care and reproductive behaviours. Recent advances in sequencing techniques have begun to elucidate the molecular basis of queen-worker differentiation. However, current knowledge of the molecular basis of caste differentiation is limited, especially to species with morphological castes. It seems likely that at the dawn of eusociality morphologically undifferentiated, monomorphic females underwent physiological differentiation that yielded egg-laying and caretaking castes. The molecular basis of such physiological differentiation may provide evolutionary insight into the emergent state of eusociality. In this study, we identify genes associated with monomorphic caste differentiation, specifically focusing on the onset of queen-worker differentiation, using a monomorphic queenless ant, Diacamma sp., that secondarily lost morphological castes. Using individuals experimentally manipulated to become sterile or reproductive, we identified 1546 caste-biased transcripts in brain and 10 in gaster. Because caste differentiation occurs in Diacamma soon after eclosion via behavioural dominance, identified transcripts are interpreted as molecular agents responding immediately to dominance rank formation. Among identified genes, expression levels of genes involved in nutrition processing and storage, such as insulin signalling genes and hexamerins, were strongly altered soon after dominance rank formation. We conclude that the rapid modification of nutrition-related genes in response to social rank may be the fundamental mechanism underlying caste differentiation in Diacamma. Together with functional evidence from the literature, we show that a specific set of genes frequently plays a role in reproductive differentiation across systems with and without morphological castes.