DEN-IM: dengue virus genotyping from amplicon and shotgun metagenomic sequencing.

Dengue virus (DENV) represents a public health threat and economic burden in affected countries. The availability of genomic data is key to understanding viral evolution and dynamics, supporting improved control strategies. Currently, the use of high-throughput sequencing (HTS) technologies, which can be applied both directly to patient samples (shotgun metagenomics) and to PCR-amplified viral sequences (amplicon sequencing), is potentially the most informative approach to monitor viral dissemination and genetic diversity by providing, in a single methodological step, identification and characterization of the whole viral genome at the nucleotide level. Despite many advantages, these technologies require bioinformatics expertise and appropriate infrastructure for the analysis and interpretation of the resulting data. In addition, the many software solutions available can hamper the reproducibility and comparison of results. Here we present DEN-IM, a one-stop, user-friendly, containerized and reproducible workflow for the analysis of DENV short-read sequencing data from both amplicon and shotgun metagenomics approaches. It is able to infer the DENV coding sequence (CDS), identify the serotype and genotype, and generate a phylogenetic tree. It can easily be run on any UNIX-like system, from local machines to high-performance computing clusters, performing a comprehensive analysis without the requirement for extensive bioinformatics expertise. Using DEN-IM, we successfully analysed two types of DENV datasets. The first comprised 25 shotgun metagenomic sequencing samples from patients with variable serotypes and genotypes, including an in vitro spiked sample containing the four known serotypes. The second consisted of 106 paired-end and 76 single-end amplicon sequences of DENV 3 genotype III and DENV 1 genotype I, respectively, where DEN-IM allowed detection of the intra-genotype diversity. The DEN-IM workflow, parameters and execution configuration files, and documentation are freely available at https://github.com/B-UMMI/DEN-IM).

Plasmid ATLAS: plasmid visual analytics and identification in high-throughput sequencing data.

Plasmid ATLAS (pATLAS, http://www.patlas.site) provides an easy-to-use web accessible database with visual analytics tools to explore the relationships of plasmids available in NCBI's RefSeq database. pATLAS has two main goals: (i) to provide an easy way to search for plasmids deposited in NCBI RefSeq and their associated metadata; (ii) to visualize the relationships of plasmids in a graph, allowing the exploration of plasmid evolution. pATLAS allows searching by plasmid name, bacterial host taxa, antibiotic resistance and virulence genes, plasmid families, and by sequence length and similarity. pATLAS is also able to represent in the plasmid network, plasmid sets identified by external pipelines using mapping, mash screen or assembly from high-throughput sequencing data. By representing the identified hits within the network of relationships between plasmids, allowing the possibility of removing redundant results, and by taking advantage of the browsing capabilities of pATLAS, users can more easily interpret the pipelines' results. All these analyses can be saved to a JSON file for sharing and future re-evaluation. Furthermore, by offering a REST-API, the pATLAS database and network display are easily accessible by other interfaces or pipelines.

chewBBACA: A complete suite for gene-by-gene schema creation and strain identification.

Gene-by-gene approaches are becoming increasingly popular in bacterial genomic epidemiology and outbreak detection. However, there is a lack of open-source scalable software for schema definition and allele calling for these methodologies. The chewBBACA suite was designed to assist users in the creation and evaluation of novel whole-genome or core-genome gene-by-gene typing schemas and subsequent allele calling in bacterial strains of interest. chewBBACA performs the schema creation and allele calls on complete or draft genomes resulting from de novo assemblers. The chewBBACA software uses Python 3.4 or higher and can run on a laptop or in high performance clusters making it useful for both small laboratories and large reference centers. ChewBBACA is available at https://github.com/B-UMMI/chewBBACA.

Structure_threader: An improved method for automation and parallelization of programs structure, fastStructure and MavericK on multicore CPU systems.

Structure_threader is a program to parallelize multiple runs of genetic clustering software that does not make use of multithreading technology (structure, fastStructure and MavericK) on multicore computers. Our approach was benchmarked across multiple systems and displayed great speed improvements relative to the single-threaded implementation, scaling very close to linearly with the number of physical cores used. Structure_threader was compared to previous software written for the same task-ParallelStructure and StrAuto and was proven to be the faster (up to 25% faster) wrapper under all tested scenarios. Furthermore, Structure_threader can perform several automatic and convenient operations, assisting the user in assessing the most biologically likely value of 'K' via implementations such as the "Evanno," or "Thermodynamic Integration" tests and automatically draw the "meanQ" plots (static or interactive) for each value of K (or even combined plots). Structure_threader is written in python 3 and licensed under the GPLv3. It can be downloaded free of charge at https://github.com/StuntsPT/Structure_threader.

The coffee leaf rust pathogen Hemileia vastatrix: one and a half centuries around the tropics.

TAXONOMY AND HISTORY: Hemileia vastatrix Berk. and Broome (Basidiomycota, Pucciniales) was described in 1869 in eastern Africa and Ceylon as the agent of coffee leaf rust and has spread to all coffee cultivation areas worldwide. Major disease outbreaks in Asia, Africa and America caused and continue to cause severe yield losses, making this the most important disease of Arabica coffee, a cash crop for many tropical and sub-tropical countries. LIFE CYCLE AND DISEASE SYMPTOMS: Hemileia vastatrix is a hemicyclic fungus with the urediniosporic life cycle as its most important (if not only) source of inoculum. Chlorotic spots are the first macroscopic symptoms, preceding the differentiation of suprastomatal, bouquet-shaped, orange-coloured uredinia. The disease can cause yield losses of up to 35% and have a polyetic epidemiological impact on subsequent years. DISEASE CONTROL: Although the use of fungicides is one of the preferred immediate control measures, the use of resistant cultivars is considered to be the most effective and durable disease control strategy. The discovery of 'Híbrido de Timor' provided sources of resistance that have been used in several breeding programmes and that have been proven to be effective and durable, as some have been in use for more than 30 years. GENETIC DIVERSITY AND MOLECULAR PATHOGENICITY: Although exhibiting limited genetic polymorphism, the very large genome of H. vastatrix (c. 797 Mbp) conceals great pathological diversity, with more than 50 physiological races. Gene expression studies have revealed a very precocious activation of signalling pathways and production of putative effectors, suggesting that the plant-fungus dialogue starts as early as at the germ tube stage, and have provided clues for the identification of avr genes.

Evolutionary and Biogeographic Insights on the Macaronesian Beta-Patellifolia Species (Amaranthaceae) from a Time-Scaled Molecular Phylogeny.

The Western Mediterranean Region and Macaronesian Islands are one of the top biodiversity hotspots of Europe, containing a significant native genetic diversity of global value among the Crop Wild Relatives (CWR). Sugar beet is the primary crop of the genus Beta (subfamily Betoideae, Amaranthaceae) and despite the great economic importance of this genus, and of the close relative Patellifolia species, a reconstruction of their evolutionary history is still lacking. We analyzed nrDNA (ITS) and cpDNA gene (matK, trnH-psbA, trnL intron, rbcL) sequences to: (i) investigate the phylogenetic relationships within the Betoideae subfamily, and (ii) elucidate the historical biogeography of wild beet species in the Western Mediterranean Region, including the Macaronesian Islands. The results support the Betoideae as a monophyletic group (excluding the Acroglochin genus) and provide a detailed inference of relationships within this subfamily, revealing: (i) a deep genetic differentiation between Beta and Patellifolia species, which may have occurred in Late Oligocene; and (ii) the occurrence of a West-East genetic divergence within Beta, indicating that the Mediterranean species probably differentiated by the end of the Miocene. This was interpreted as a signature of species radiation induced by dramatic habitat changes during the Messinian Salinity Crisis (MSC, 5.96-5.33 Mya). Moreover, colonization events during the Pleistocene also played a role in shaping the current diversity patterns among and within the Macaronesian Islands. The origin and number of these events could not be revealed due to insufficient phylogenetic resolution, suggesting that the diversification was quite recent in these archipelagos, and unravelling potential complex biogeographic patterns with hybridization and gene flow playing an important role. Finally, three evolutionary lineages were identified corresponding to major gene pools of sugar beet wild relatives, which provide useful information for establishing in situ and ex situ conservation priorities in the hotspot area of the Macaronesian Islands.

Genomic Patterns of Positive Selection at the Origin of Rust Fungi.

Understanding the origin and evolution of pathogenicity and biotrophic life-style of rust fungi has remained a conundrum for decades. Research on the molecular mechanisms responsible for rust fungi evolution has been hampered by their biotrophic life-style until the sequencing of some rust fungi genomes. With the availability of multiple whole genomes and EST data for this group, it is now possible to employ genome-wide surveys and investigate how natural selection shaped their evolution. In this work, we employed a phylogenomics approach to search for positive selection and genes undergoing accelerated evolution at the origin of rust fungi on an assembly of single copy genes conserved across a broad range of basidiomycetes. Up to 985 genes were screened for positive selection on the phylogenetic branch leading to rusts, revealing a pervasive signal of positive selection throughout the data set with the proportion of positively selected genes ranging between 19.6-33.3%. Additionally, 30 genes were found to be under accelerated evolution at the origin of rust fungi, probably due to a mixture of positive selection and relaxation of purifying selection. Functional annotation of the positively selected genes revealed an enrichment in genes involved in the biosynthesis of secondary metabolites and several metabolism and transporter classes.

New mitochondrial and nuclear evidences support recent demographic expansion and an atypical phylogeographic pattern in the spittlebug Philaenus spumarius (Hemiptera, Aphrophoridae).

Philaenus spumarius is a widespread insect species in the Holarctic region. Here, by focusing on the mtDNA gene COI but also using the COII and Cyt b genes and the nuclear gene EF-1α, we tried to explain how and when its current biogeographic pattern evolved by providing time estimates of the main demographic and evolutionary events and investigating its colonization patterns in and out of Eurasia. Evidence of recent divergence and expansion events at less than 0.5 Ma ago indicate that climate fluctuations in the Mid-Late Pleistocene were important in shaping the current phylogeographic pattern of the species. Data support a first split and differentiation of P. spumarius into two main mitochondrial lineages: the "western", in the Mediterranean region and the "eastern", in Anatolia/Caucasus. It also supports a following differentiation of the "western" lineage into two sub-lineages: the "western-Mediterranean", in Iberia and the "eastern-Mediterranean" in the Balkans. The recent pattern seems to result from postglacial range expansion from Iberia and Caucasus/Anatolia, thus not following one of the four common paradigms. Unexpected patterns of recent gene-flow events between Mediterranean peninsulas, a close relationship between Iberia and North Africa, as well as high levels of genetic diversity being maintained in northern Europe were found. The mitochondrial pattern does not exactly match to the nuclear pattern suggesting that the current biogeographic pattern of P. spumarius may be the result of both secondary admixture and incomplete lineage sorting. The hypothesis of recent colonization of North America from both western and northern Europe is corroborated by our data and probably resulted from accidental human translocations. A probable British origin for the populations of the Azores and New Zealand was revealed, however, for the Azores the distribution of populations in high altitude native forests is somewhat puzzling and may imply a natural colonization of the archipelago.

Host-jump drives rapid and recent ecological speciation of the emergent fungal pathogen Colletotrichum kahawae.

Ecological speciation through host-shift has been proposed as a major route for the appearance of novel fungal pathogens. The growing awareness of their negative impact on global economies and public health created an enormous interest in identifying the factors that are most likely to promote their emergence in nature. In this work, a combination of pathological, molecular and geographical data was used to investigate the recent emergence of the fungus Colletotrichum kahawae. C. kahawae emerged as a specialist pathogen causing coffee berry disease in Coffea arabica, owing to its unparalleled adaptation of infecting green coffee berries. Contrary to current hypotheses, our results suggest that a recent host-jump underlay the speciation of C. kahawae from a generalist group of fungi seemingly harmless to coffee berries. We posit that immigrant inviability and a predominantly asexual behaviour could have been instrumental in driving speciation by creating pleiotropic interactions between local adaptation and reproductive patterns. Moreover, we estimate that C. kahawae began its diversification at <2200 bp leaving a very short time frame since the divergence from its sibling lineage (c. 5600 bp), during which a severe drop in C. kahawae's effective population size occurred. This further supports a scenario of recent introduction and subsequent adaptation to C. arabica. Phylogeographical data revealed low levels of genetic polymorphism but provided the first geographically consistent population structure of C. kahawae, inferring the Angolan population as the most ancestral and the East African populations as the most recently derived. Altogether, these results highlight the significant role of host specialization and asexuality in the emergence of fungal pathogens through ecological speciation.