Generating the raw data for a de novo genome assembly project for a target eukaryotic species is relatively easy. This democratization of access to large-scale data has allowed many research teams to plan to assemble the genomes of non-model organisms. These new genome targets are very different from the traditional, inbred, laboratory-reared model organisms. They are often small, and cannot be isolated free of their environment – whether ingested food, the surrounding host organism of parasites, or commensal and symbiotic organisms attached to or within the individuals sampled. Preparation of pure DNA originating from a single species can be technically impossible, but assembly of mixed-organism DNA can be difficult, as most genome assemblers perform poorly when faced with multiple genomes in different stoichiometries. This class of problem is common in metagenomic datasets that deliberately try to capture all the genomes present in an environment, but replicon assembly is not often the goal of such programs. Here we present an approach to extracting, from mixed DNA sequence data, subsets that correspond to single species’ genomes and thus improving genome assembly. We use both numerical (proportion of GC bases and read coverage) and biological (best-matching sequence in annotated databases) indicators to aid partitioning of draft assembly contigs, and the reads that contribute to those contigs, into distinct bins that can then be subjected to rigorous, optimized assembly, through the use of taxon-annotated GC-coverage plots (TAGC plots). We also present Blobsplorer, a tool that aids exploration and selection of subsets from TAGC-annotated data. Partitioning the data in this way can rescue poorly assembled genomes, and reveal unexpected symbionts and commensals in eukaryotic genome projects. The TAGC plot pipeline script is available from https://github.com/blaxterlab/blobology, and the Blobsplorer tool from https://github.com/mojones/Blobsplorer.

SUMMARY: Nematodes are abundant and diverse, and include many parasitic species. Molecular phylogenetic analyses have shown that parasitism of plants and animals has arisen at least 15 times independently. Extant nematode species also display lifestyles that are proposed to be on the evolutionary trajectory to parasitism. Recent advances have permitted the determination of the genomes and transcriptomes of many nematode species. These new data can be used to further resolve the phylogeny of Nematoda, and identify possible genetic patterns associated with parasitism. Plant-parasitic nematode genomes show evidence of horizontal gene transfer from other members of the rhizosphere, and these genes play important roles in the parasite-host interface. Similar horizontal transfer is not evident in animal parasitic groups. Many nematodes have bacterial symbionts that can be essential for survival. Horizontal transfer from symbionts to the nematode is also common, but its biological importance is unclear. Over 100 nematode species are currently targeted for sequencing, and these data will yield important insights into the biology and evolutionary history of parasitism. It is important that these new technologies are also applied to free-living taxa, so that the pre-parasitic ground state can be inferred, and the novelties associated with parasitism isolated.

Significance: Tardigrades, also known as moss piglets or water bears, are renowned for their ability to withstand extreme environmental challenges. A recently published analysis of the genome of the tardigrade Hypsibius dujardini by Boothby et al. concluded that horizontal acquisition of genes from bacterial and other sources might be key to cryptobiosis in tardigrades. We independently sequenced the genome of H. dujardini and detected a low level of horizontal gene transfer. We show that the extensive horizontal transfer proposed by Boothby et al. was an artifact of a failure to eliminate contaminants from sequence data before assembly.

Tardigrada, a phylum of meiofaunal organisms, have been at the center of discussions of the evolution of Metazoa, the biology of survival in extreme environments, and the role of horizontal gene transfer in animal evolution. Tardigrada are placed as sisters to Arthropoda and Onychophora (velvet worms) in the superphylum Panarthropoda by morphological analyses, but many molecular phylogenies fail to recover this relationship. This tension between molecular and morphological understanding may be very revealing of the mode and patterns of evolution of major groups. Limnoterrestrial tardigrades display extreme cryptobiotic abilities, including anhydrobiosis and cryobiosis, as do bdelloid rotifers, nematodes, and other animals of the water film. These extremophile behaviors challenge understanding of normal, aqueous physiology: how does a multicellular organism avoid lethal cellular collapse in the absence of liquid water? Meiofaunal species have been reported to have elevated levels of horizontal gene transfer (HGT) events, but how important this is in evolution, and particularly in the evolution of extremophile physiology, is unclear. To address these questions, we resequenced and reassembled the genome of H. dujardini, a limnoterrestrial tardigrade that can undergo anhydrobiosis only after extensive pre-exposure to drying conditions, and compared it to the genome of R. varieornatus, a related species with tolerance to rapid desiccation. The 2 species had contrasting gene expression responses to anhydrobiosis, with major transcriptional change in H. dujardini but limited regulation in R. varieornatus. We identified few horizontally transferred genes, but some of these were shown to be involved in entry into anhydrobiosis. Whole-genome molecular phylogenies supported a Tardigrada+Nematoda relationship over Tardigrada+Arthropoda, but rare genomic changes tended to support Tardigrada+Arthropoda. Tardigrades are justly famous for their abilities to withstand environmental extremes. Many freshwater and terrestrial species can undergo anhydrobiosis—life without water—and thereby withstand desiccation, freezing, and other insults. We explored the comparative biology of anhydrobiosis in 2 species of tardigrade that differ in the mechanisms they use to enter anhydrobiosis. Using newly assembled and improved genomes, we find that Ramazzottius varieornatus, a species that can withstand rapid desiccation, differs from Hypsibius dujardini, a species that requires extended preconditioning, in not showing a major transcriptional response to anhydrobiosis induction. We identified a number of genetic systems in the tardigrades that likely play conserved, central roles in anhydrobiosis as well as species-unique components. Compared to previous estimates, our improved genomes show much reduced levels of horizontal gene transfer into tardigrade genomes, but some of the identified horizontal gene transfer (HGT) genes appear to be involved in anhydrobiosis. Using the improved genomes, we explored the evolutionary relationships of tardigrades and other molting animals, particularly nematodes and arthropods. We identified conflicting signals between sequence-based analyses, which found a relationship between tardigrades and nematodes, and analyses based on rare genomic changes, which tended to support the traditional tardigrade-arthropod link.

While components of the pathway that establishes left-right asymmetry have been identified in diverse animals, from vertebrates to flies, it is striking that the genes involved in the first symmetry-breaking step remain wholly unknown in the most obviously chiral animals, the gastropod snails. Previously, research on snails was used to show that left-right signaling of Nodal, downstream of symmetry breaking, may be an ancestral feature of the Bilateria [1 and 2]. Here, we report that a disabling mutation in one copy of a tandemly duplicated, diaphanous-related formin is perfectly associated with symmetry breaking in the pond snail. This is supported by the observation that an anti-formin drug treatment converts dextral snail embryos to a sinistral phenocopy, and in frogs, drug inhibition or overexpression by microinjection of formin has a chirality-randomizing effect in early (pre-cilia) embryos. Contrary to expectations based on existing models [3, 4 and 5], we discovered asymmetric gene expression in 2- and 4-cell snail embryos, preceding morphological asymmetry. As the formin-actin filament has been shown to be part of an asymmetry-breaking switch in vitro [6 and 7], together these results are consistent with the view that animals with diverse body plans may derive their asymmetries from the same intracellular chiral elements [8].

The yellow potato cyst nematode, Globodera rostochiensis, is a devastating plant pathogen of global economic importance. This biotrophic parasite secretes effectors from pharyngeal glands, some of which were acquired by horizontal gene transfer, to manipulate host processes and promote parasitism. G. rostochiensis is classified into pathotypes with different plant resistance-breaking phenotypes. We generate a high quality genome assembly for G. rostochiensis pathotype Ro1, identify putative effectors and horizontal gene transfer events, map gene expression through the life cycle focusing on key parasitic transitions and sequence the genomes of eight populations including four additional pathotypes to identify variation. Horizontal gene transfer contributes 3.5 % of the predicted genes, of which approximately 8.5 % are deployed as effectors. Over one-third of all effector genes are clustered in 21 putative ‘effector islands’ in the genome. We identify a dorsal gland promoter element motif (termed DOG Box) present upstream in representatives from 26 out of 28 dorsal gland effector families, and predict a putative effector superset associated with this motif. We validate gland cell expression in two novel genes by in situ hybridisation and catalogue dorsal gland promoter element-containing effectors from available cyst nematode genomes. Comparison of effector diversity between pathotypes highlights correlation with plant resistance-breaking. These G. rostochiensis genome resources will facilitate major advances in understanding nematode plant-parasitism. Dorsal gland promoter element-containing effectors are at the front line of the evolutionary arms race between plant and parasite and the ability to predict gland cell expression a priori promises rapid advances in understanding their roles and mechanisms of action.