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Characterization of Human Endogenous Retrovirus (HERV) expression within the transcriptomic landscape using RNA-seq is complicated by uncertainty in fragment assignment because of sequence similarity. We present Telescope, a computational software tool that provides accurate estimation of transposable element expression (retrotranscriptome) resolved to specific genomic locations. Telescope directly addresses uncertainty in fragment assignment by reassigning ambiguously mapped fragments to the most probable source transcript as determined within a Bayesian statistical model. We demonstrate the utility of our approach through single locus analysis of HERV expression in 13 ENCODE cell types. When examined at this resolution, we find that the magnitude and breadth of the retrotranscriptome can be vastly different among cell types. Furthermore, our approach is robust to differences in sequencing technology and demonstrates that the retrotranscriptome has potential to be used for cell type identification. We compared our tool with other approaches for quantifying transposable element (TE) expression, and found that Telescope has the greatest resolution, as it estimates expression at specific TE insertions rather than at the TE subfamily level. Telescope performs highly accurate quantification of the retrotranscriptomic landscape in RNA-seq experiments, revealing a differential complexity in the transposable element biology of complex systems not previously observed. Telescope is available at https://github.com/mlbendall/telescope., Competing Interests: The authors have declared that no competing interests exist.

A comprehensive knowledge of the types and ratios of microbes that inhabit the healthy human gut is necessary before any kind of pre-clinical or clinical study can be performed that attempts to alter the microbiome to treat a condition or improve therapy outcome. To address this need we present an innovative scalable comprehensive analysis workflow, a healthy human reference microbiome list and abundance profile (GutFeelingKB), and a novel Fecal Biome Population Report (FecalBiome) with clinical applicability. GutFeelingKB provides a list of 157 organisms (8 phyla, 18 classes, 23 orders, 38 families, 59 genera and 109 species) that forms the baseline biome and therefore can be used as healthy controls for studies related to dysbiosis. This list can be expanded to 863 organisms if closely related proteomes are considered. The incorporation of microbiome science into routine clinical practice necessitates a standard report for comparison of an individual's microbiome to the growing knowledgebase of "normal" microbiome data. The FecalBiome and the underlying technology of GutFeelingKB address this need. The knowledgebase can be useful to regulatory agencies for the assessment of fecal transplant and other microbiome products, as it contains a list of organisms from healthy individuals. In addition to the list of organisms and their abundances, this study also generated a collection of assembled contiguous sequences (contigs) of metagenomics dark matter. In this study, metagenomic dark matter represents sequences that cannot be mapped to any known sequence but can be assembled into contigs of 10,000 nucleotides or higher. These sequences can be used to create primers to study potential novel organisms. All data is freely available from https://hive.biochemistry.gwu.edu/gfkb and NCBI's Short Read Archive., Competing Interests: The authors have declared that no competing interests exist.

Background: Washington DC has a high burden of HIV with a 2.0% HIV prevalence. The city is a national and international hub potentially containing a broad diversity of HIV variants; yet few sequences from DC are available on GenBank to assess the evolutionary history of HIV in the US capital. Towards this general goal, here we analyze extensive sequence data and investigate HIV diversity, phylodynamics, and drug resistant mutations (DRM) in DC., Methods: Molecular HIV-1 sequences were collected from participants infected through 2015 as part of the DC Cohort, a longitudinal observational study of HIV+ patients receiving care at 13 DC clinics. Sequences were paired with Cohort demographic, risk, and clinical data and analyzed using maximum likelihood, Bayesian and coalescent approaches of phylogenetic, network and population genetic inference. We analyzed 601 sequences from 223 participants for int (~864 bp) and 2,810 sequences from 1,659 participants for PR/RT (~1497 bp)., Results: Ninety-nine and 94% of the int and PR/RT sequences, respectively, were identified as subtype B, with 14 non-B subtypes also detected. Phylodynamic analyses of US born infected individuals showed that HIV population size varied little over time with no significant decline in diversity. Phylogenetic analyses grouped 13.5% of the int sequences into 14 clusters of 2 or 3 sequences, and 39.0% of the PR/RT sequences into 203 clusters of 2-32 sequences. Network analyses grouped 3.6% of the int sequences into 4 clusters of 2 sequences, and 10.6% of the PR/RT sequences into 76 clusters of 2-7 sequences. All network clusters were detected in our phylogenetic analyses. Higher proportions of clustered sequences were found in zip codes where HIV prevalence is highest (r = 0.607; P<0.00001). We detected a high prevalence of DRM for both int (17.1%) and PR/RT (39.1%), but only 8 int and 12 PR/RT amino acids were identified as under adaptive selection. We observed a significant (P<0.0001) association between main risk factors (men who have sex with men and heterosexuals) and genotypes in the five well-supported clusters with sufficient sample size for testing., Discussion: Pairing molecular data with clinical and demographic data provided novel insights into HIV population dynamics in Washington, DC. Identification of populations and geographic locations where clustering occurs can inform and complement active surveillance efforts to interrupt HIV transmission.

Background: The nasopharynx is a reservoir for pathogens associated with respiratory illnesses such as asthma. Next-generation sequencing (NGS) has been used to characterize the nasopharyngeal microbiome of infants and adults during health and disease; less is known, however, about the composition and temporal dynamics (i.e., longitudinal variation) of microbiotas from children and adolescents. Here we use NGS technology to characterize the nasopharyngeal microbiomes of asthmatic children and adolescents (6 to 18 years) and determine their stability over time., Methods: Two nasopharyngeal washes collected 5.5 to 6.5 months apart were taken from 40 children and adolescents with asthma living in the Washington D.C. area. Sequence data from the 16S-V4 rRNA gene region (~250 bp) were collected from the samples using the MiSeq platform. Raw data were processed in mothur (SILVA123 reference database) and Operational Taxonomic Units (OTU)-based alpha- and beta-diversity metrics were estimated. Relatedness among samples was assessed using PCoA ordination and Procrustes analyses. Differences in microbial diversity and taxon mean relative proportions were assessed using linear mixed effects models. Core microbiome analyses were also performed to identify stable and consistent microbes of the nasopharynx., Results and Discussion: A total of 2,096,584 clean 16S sequences corresponding to an average of 167 OTUs per sample were generated. Representatives of Moraxella*, Staphylococcus*, Dolosigranulum, Corynebacterium, Prevotella, Streptococcus*, Haemophilus*, Fusobacterium* and a Neisseriaceae genus accounted for 86% of the total reads. These nine genera have been previously found in the nasopharynxes of both infants and adults, but in different proportions. OTUs from the five genera highlighted (*) above defined the nasopharyngeal core microbiome at the 95% level. No significant differences in alpha- and beta-diversity were observed between seasons, but bacterial mean relative proportions of Haemophilus, Moraxella, Staphylococcus and Corynebacterium varied significantly between summer-fall and age groups (inter-patient variation). Additionally, OTUs varied significantly within patients between time points in 35 of the 40 patients analyzed. Future cross-sectional studies should be mindful of the temporal dynamics of the nasopharyngeal microbiota., Competing Interests: The authors have declared that no competing interests exist.

Background: High-throughput sequencing (HTS) analysis of microbial communities from the respiratory airways has heavily relied on the 16S rRNA gene. Given the intrinsic limitations of this approach, airway microbiome research has focused on assessing bacterial composition during health and disease, and its variation in relation to clinical and environmental factors, or other microbiomes. Consequently, very little effort has been dedicated to describing the functional characteristics of the airway microbiota and even less to explore the microbe-host interactions. Here we present a simultaneous assessment of microbiome and host functional diversity and host-microbe interactions from the same RNA-seq experiment, while accounting for variation in clinical metadata., Methods: Transcriptomic (host) and metatranscriptomic (microbiota) sequences from the nasal epithelium of 8 asthmatics and 6 healthy controls were separated in silico and mapped to available human and NCBI-NR protein reference databases. Human genes differentially expressed in asthmatics and controls were then used to infer upstream regulators involved in immune and inflammatory responses. Concomitantly, microbial genes were mapped to metabolic databases (COG, SEED, and KEGG) to infer microbial functions differentially expressed in asthmatics and controls. Finally, multivariate analysis was applied to find associations between microbiome characteristics and host upstream regulators while accounting for clinical variation., Results and Discussion: Our study showed significant differences in the metabolism of microbiomes from asthmatic and non-asthmatic children for up to 25% of the functional properties tested. Enrichment analysis of 499 differentially expressed host genes for inflammatory and immune responses revealed 43 upstream regulators differentially activated in asthma. Microbial adhesion (virulence) and Proteobacteria abundance were significantly associated with variation in the expression of the upstream regulator IL1A; suggesting that microbiome characteristics modulate host inflammatory and immune systems during asthma.

Competing Interests: The authors have declared that no competing interests exist.

The genomic island encoding the locus of enterocyte effacement (LEE) is an important virulence factor of the human pathogenic Escherichia coli. LEE typically encodes a type III secretion system (T3SS) and secreted effectors capable of forming attaching and effacing lesions. Although prominent in the pathogenic E. coli such as serotype O157:H7, LEE has also been detected in Citrobacter rodentium, E. albertii, and although not confirmed, it is likely to also be in Shigella boydii. Previous phylogenetic analysis of LEE indicated the genomic island was evolving through stepwise acquisition of various components. This study describes a new LEE region from two strains of Salmonella enterica subspecies salamae serovar Sofia along with a phylogenetic analysis of LEE that provides new insights into the likely evolution of this genomic island. The Salmonella LEE contains 36 of the 41 genes typically observed in LEE within a genomic island of 49, 371 bp that encodes a total of 54 genes. A phylogenetic analysis was performed on the entire T3SS and four T3SS genes (escF, escJ, escN, and escV) to elucidate the genealogy of LEE. Phylogenetic analysis inferred that the previously known LEE islands are members of a single lineage distinct from the new Salmonella LEE lineage. The previously known lineage of LEE diverged between islands found in Citrobacter and those in Escherichia and Shigella. Although recombination and horizontal gene transfer are important factors in the genealogy of most genomic islands, the phylogeny of the T3SS of LEE can be interpreted with a bifurcating tree. It seems likely that the LEE island entered the Enterobacteriaceae through horizontal gene transfer as a single unit, rather than as separate subsections, which was then subjected to the forces of both mutational change and recombination.

Background: Multiple loci and population genetic methods were employed to study the phylogeographic history of the Patagonian freshwater crab Aegla neuquensis (Aeglidae: Decopoda). This taxon occurs in two large river systems in the Patagonian Steppe, from the foothills of the Andes Mountains east to the Atlantic Ocean., Methodology/principal Findings: A nuclear phylogeny and multilocus nested clade phylogeographic analysis detected a fragmentation event between the Negro and Chico-Chubut river systems. This event occurred approximately 137 thousand years ago. An isolation-with-migration analysis and maximum-likelihood estimates of gene flow showed asymmetrical exchange of genetic material between these two river systems exclusively in their headwaters. We used information theory to determine the best-fit demographic history between these two river systems under an isolation-with-migration model. The best-fit model suggests that the Negro and the ancestral populations have the same effective population sizes; whereas the Chico-Chubut population is smaller and shows that gene flow from the Chico-Chubut into the Negro is four times higher than in the reverse direction. Much of the Chico-Chubut system appears to have only been recently colonized while the Negro populations appear to have been in place for most of the evolutionary history of this taxon., Conclusions/significance: Due to mitochondrial introgression, three nuclear loci provided different phylogeographic resolution than the three mitochondrial genes for an ancient fragmentation event observed in the nuclear phylogeny. However, the mitochondrial locus provided greater resolution on more recent evolutionary events. Our study, therefore, demonstrates the need to include both nuclear and mitochondrial loci for a more complete understanding of evolutionary histories and associated phylogeographic events. Our results suggest that gene flow between these systems, before and after fragmentation was through periodic paleolakes that formed in the headwaters region. Fragmentation between the Negro and Chico-Chubut systems was driven by the disappearance of these paleolakes during the Patagonian Glaciation.

Background: The genus Cambarus is one of three most species rich crayfish genera in the Northern Hemisphere. The genus has its center of diversity in the Southern Appalachians of the United States and has been divided into 12 subgenera. Using Cambarus we test the correspondence of subgeneric designations based on morphology used in traditional crayfish taxonomy to the underlying evolutionary history for these crayfish. We further test for significant correlation and explanatory power of geographic distance, taxonomic model, and a habitat model to estimated phylogenetic distance with multiple variable regression., Methodology/principal Findings: We use three mitochondrial and one nuclear gene regions to estimate the phylogenetic relationships for species within the genus Cambarus and test evolutionary hypotheses of relationships and associated morphological and biogeographical hypotheses. Our resulting phylogeny indicates that the genus Cambarus is polyphyletic, however we fail to reject the monophyly of Cambarus with a topology test. The majority of the Cambarus subgenera are rejected as monophyletic, suggesting the morphological characters used to define those taxa are subject to convergent evolution. While we found incongruence between taxonomy and estimated phylogenetic relationships, a multiple model regression analysis indicates that taxonomy had more explanatory power of genetic relationships than either habitat or geographic distance., Conclusions: We find convergent evolution has impacted the morphological features used to delimit Cambarus subgenera. Studies of the crayfish genus Orconectes have shown gonopod morphology used to delimit subgenera is also affected by convergent evolution. This suggests that morphological diagnoses based on traditional crayfish taxonomy might be confounded by convergent evolution across the cambarids and has little utility in diagnosing relationships or defining natural groups. We further suggest that convergent morphological evolution appears to be a common occurrence in invertebrates suggesting the need for careful phylogenetically based interpretations of morphological evolution in invertebrate systematics.

The Dwarf crayfish or Cambarellinae, is a morphologically singular subfamily of decapod crustaceans that contains only one genus, Cambarellus. Its intriguing distribution, along the river basins of the Gulf Coast of United States (Gulf Group) and into Central México (Mexican Group), has until now lacked of satisfactory explanation. This study provides a comprehensive sampling of most of the extant species of Cambarellus and sheds light on its evolutionary history, systematics and biogeography. We tested the impact of Gulf Group versus Mexican Group geography on rates of cladogenesis using a maximum likelihood framework, testing different models of birth/extinction of lineages. We propose a comprehensive phylogenetic hypothesis for the subfamily based on mitochondrial and nuclear loci (3,833 bp) using Bayesian and Maximum Likelihood methods. The phylogenetic structure found two phylogenetic groups associated to the two main geographic components (Gulf Group and Mexican Group) and is partially consistent with the historical structure of river basins. The previous hypothesis, which divided the genus into three subgenera based on genitalia morphology was only partially supported (P = 0.047), resulting in a paraphyletic subgenus Pandicambarus. We found at least two cases in which phylogenetic structure failed to recover monophyly of recognized species while detecting several cases of cryptic diversity, corresponding to lineages not assigned to any described species. Cladogenetic patterns in the entire subfamily are better explained by an allopatric model of speciation. Diversification analyses showed similar cladogenesis patterns between both groups and did not significantly differ from the constant rate models. While cladogenesis in the Gulf Group is coincident in time with changes in the sea levels, in the Mexican Group, cladogenesis is congruent with the formation of the Trans-Mexican Volcanic Belt. Our results show how similar allopatric divergence in freshwater organisms can be promoted through diverse vicariant factors.

Background: In 2003, a phase III placebo-controlled trial (VAX003) was completed in Bangkok, Thailand. Of the 2,546 individuals enrolled in the trial based on high risk for infection through injection drug use (IDU), we obtained clinical samples and HIV-1 sequence data (envelope glycoprotein gene gp120) from 215 individuals who became infected during the trial. Here, we used these data in combination with other publicly available gp120 sequences to perform a molecular surveillance and phylodynamic analysis of HIV-1 in Thailand., Methodology and Findings: Phylogenetic and population genetic estimators were used to assess HIV-1 gp120 diversity as a function of vaccination treatment, viral load (VL) and CD4(+) counts, to identify transmission clusters and to investigate the timescale and demographics of HIV-1 in Thailand. Three HIV-1 subtypes were identified: CRF01&#95;AE (85% of the infections), subtype B (13%) and CRF15&#95;AE (2%). The Bangkok IDU cohort showed more gp120 diversity than other Asian IDU cohorts and similar diversity to that observed in sexually infected individuals. Moreover, significant differences (P<0.02) in genetic diversity were observed in CRF01&#95;AE IDU with different VL and CD4(+) counts. No phylogenetic structure was detected regarding any of the epidemiological and clinical factors tested, although high proportions (35% to 50%) of early infections fell into clusters, which suggests that transmission chains associated with acute infection play a key role on HIV-1 spread among IDU. CRF01&#95;AE was estimated to have emerged in Thailand in 1984.5 (1983-1986), 3-6 years before the first recognition of symptomatic patients (1989). The relative genetic diversity of the HIV-1 population has remained high despite decreasing prevalence rates since the mid 1990s., Conclusions: Our study and recent epidemiological reports indicate that HIV-1 is still a major threat in Thailand and suggest that HIV awareness and prevention needs to be strengthened to avoid AIDS resurgence.

Background: We present a series of simulation studies that explore the relative performance of several phylogenetic network approaches (statistical parsimony, split decomposition, union of maximum parsimony trees, neighbor-net, simulated history recombination upper bound, median-joining, reduced median joining and minimum spanning network) compared to standard tree approaches, (neighbor-joining and maximum parsimony) in the presence and absence of recombination., Principal Findings: In the absence of recombination, all methods recovered the correct topology and branch lengths nearly all of the time when the substitution rate was low, except for minimum spanning networks, which did considerably worse. At a higher substitution rate, maximum parsimony and union of maximum parsimony trees were the most accurate. With recombination, the ability to infer the correct topology was halved for all methods and no method could accurately estimate branch lengths., Conclusions: Our results highlight the need for more accurate phylogenetic network methods and the importance of detecting and accounting for recombination in phylogenetic studies. Furthermore, we provide useful information for choosing a network algorithm and a framework in which to evaluate improvements to existing methods and novel algorithms developed in the future.