BackgroundIn the last 5 years, the rapid pace of innovations and improvements in sequencing technologies has completely changed the landscape of metagenomic and metagenetic experiments. Therefore, it is critical to benchmark the various methodologies for interrogating the composition of microbial communities, so that we can assess their strengths and limitations. The most common phylogenetic marker for microbial community diversity studies is the 16S ribosomal RNA gene and in the last 10 years the field has moved from sequencing a small number of amplicons and samples to more complex studies where thousands of samples and multiple different gene regions are interrogated.ResultsWe assembled 2 synthetic communities with an even (EM) and uneven (UM) distribution of archaeal and bacterial strains and species, as metagenomic control material, to assess performance of different experimental strategies. The 2 synthetic communities were used in this study, to highlight the limitations and the advantages of the leading sequencing platforms: MiSeq (Illumina), The Pacific Biosciences RSII, 454 GS-FLX/+ (Roche), and IonTorrent (Life Technologies). We describe an extensive survey based on synthetic communities using 3 experimental designs (fusion primers, universal tailed tag, ligated adaptors) across the 9 hypervariable 16S rDNA regions. We demonstrate that library preparation methodology can affect data interpretation due to different error and chimera rates generated during the procedure. The observed community composition was always biased, to a degree that depended on the platform, sequenced region and primer choice. However, crucially, our analysis suggests that 16S rRNA sequencing is still quantitative, in that relative changes in abundance of taxa between samples can be recovered, despite these biases.ConclusionWe have assessed a range of experimental conditions across several next generation sequencing platforms using the most up-to-date configurations. We propose that the choice of sequencing platform and experimental design needs to be taken into consideration in the early stage of a project by running a small trial consisting of several hypervariable regions to quantify the discriminatory power of each region. We also suggest that the use of a synthetic community as a positive control would be beneficial to identify the potential biases and procedural drawbacks that may lead to data misinterpretation. The results of this study will serve as a guideline for making decisions on which experimental condition and sequencing platform to consider to achieve the best microbial profiling.Electronic supplementary materialThe online version of this article (doi:10.1186/s12864-015-2194-9) contains supplementary material, which is available to authorized users.

Analysis of 179 new Ebola virus sequences from patient samples collected in Guinea between March 2014 and January 2015 shows how different lineages evolved and spread in West Africa. Supplementary information The online version of this article (doi:10.1038/nature14594) contains supplementary material, which is available to authorized users.

BackgroundIn 2014, Western Africa experienced an unanticipated explosion of Ebola virus infections. What distinguishes fatal from non-fatal outcomes remains largely unknown, yet is key to optimising personalised treatment strategies. We used transcriptome data for peripheral blood taken from infected and convalescent recovering patients to identify early stage host factors that are associated with acute illness and those that differentiate patient survival from fatality.ResultsThe data demonstrate that individuals who succumbed to the disease show stronger upregulation of interferon signalling and acute phase responses compared to survivors during the acute phase of infection. Particularly notable is the strong upregulation of albumin and fibrinogen genes, which suggest significant liver pathology. Cell subtype prediction using messenger RNA expression patterns indicated that NK-cell populations increase in patients who survive infection. By selecting genes whose expression properties discriminated between fatal cases and survivors, we identify a small panel of responding genes that act as strong predictors of patient outcome, independent of viral load.ConclusionsTranscriptomic analysis of the host response to pathogen infection using blood samples taken during an outbreak situation can provide multiple levels of information on both disease state and mechanisms of pathogenesis. Host biomarkers were identified that provide high predictive value under conditions where other predictors, such as viral load, are poor prognostic indicators. The data suggested that rapid analysis of the host response to infection in an outbreak situation can provide valuable information to guide an understanding of disease outcome and mechanisms of disease.Electronic supplementary materialThe online version of this article (doi:10.1186/s13059-016-1137-3) contains supplementary material, which is available to authorized users.

Staphylococcus aureus is an important human commensal and opportunistic pathogen responsible for a wide range of infections. Long chain unsaturated free fatty acids represent a barrier to colonisation and infection by S. aureus and act as an antimicrobial component of the innate immune system where they are found on epithelial surfaces and in abscesses. Despite many contradictory reports, the precise anti-staphylococcal mode of action of free fatty acids remains undetermined. In this study, transcriptional (microarrays and qRT-PCR) and translational (proteomics) analyses were applied to ascertain the response of S. aureus to a range of free fatty acids. An increase in expression of the σB and CtsR stress response regulons was observed. This included increased expression of genes associated with staphyloxanthin synthesis, which has been linked to membrane stabilisation. Similarly, up-regulation of genes involved in capsule formation was recorded as were significant changes in the expression of genes associated with peptidoglycan synthesis and regulation. Overall, alterations were recorded predominantly in pathways involved in cellular energetics. In addition, sensitivity to linoleic acid of a range of defined (sigB, arcA, sasF, sarA, agr, crtM) and transposon-derived mutants (vraE, SAR2632) was determined. Taken together, these data indicate a common mode of action for long chain unsaturated fatty acids that involves disruption of the cell membrane, leading to interference with energy production within the bacterial cell. Contrary to data reported for other strains, the clinically important EMRSA-16 strain MRSA252 used in this study showed an increase in expression of the important virulence regulator RNAIII following all of the treatment conditions tested. An adaptive response by S. aureus of reducing cell surface hydrophobicity was also observed. Two fatty acid sensitive mutants created during this study were also shown to diplay altered pathogenesis as assessed by a murine arthritis model. Differences in the prevalence and clinical importance of S. aureus strains might partly be explained by their responses to antimicrobial fatty acids.

Decline-diseases are complex and becoming increasingly problematic to tree health globally. Acute Oak Decline (AOD) is characterized by necrotic stem lesions and galleries of the bark-boring beetle, Agrilus biguttatus, and represents a serious threat to oak. Although multiple novel bacterial species and Agrilus galleries are associated with AOD lesions, the causative agent(s) are unknown. The AOD pathosystem therefore provides an ideal model for a systems-based research approach to address our hypothesis that AOD lesions are caused by a polymicrobial complex. Here we show that three bacterial species, Brenneria goodwinii, Gibbsiella quercinecans and Rahnella victoriana, are consistently abundant in the lesion microbiome and possess virulence genes used by canonical phytopathogens that are expressed in AOD lesions. Individual and polyspecies inoculations on oak logs and trees demonstrated that B. goodwinii and G. quercinecans cause tissue necrosis and, in combination with A. biguttatus, produce the diagnostic symptoms of AOD. We have proved a polybacterial cause of AOD lesions, providing new insights into polymicrobial interactions and tree disease. This work presents a novel conceptual and methodological template for adapting Koch’s postulates to address the role of microbial communities in disease.

For many highly mobile species, the marine environment presents few obvious barriers to gene flow. Even so, there is considerable diversity within and among species, referred to by some as the 'marine speciation paradox'. The recent and diverse radiation of delphinid cetaceans (dolphins) represents a good example of this. Delphinids are capable of extensive dispersion and yet many show fine-scale genetic differentiation among populations. Proposed mechanisms include the division and isolation of populations based on habitat dependence and resource specializations, and habitat release or changing dispersal corridors during glacial cycles. Here we use a phylogenomic approach to investigate the origin of differentiated sympatric populations of killer whales (Orcinus orca). Killer whales show strong specialization on prey choice in populations of stable matrifocal social groups (ecotypes), associated with genetic and phenotypic differentiation. Our data suggest evolution in sympatry among populations of resource specialists.

Soil biota accounts for ~25% of global biodiversity and is vital to nutrient cycling and primary production. There is growing momentum to study total belowground biodiversity across large ecological scales to understand how habitat and soil properties shape belowground communities. Microbial and animal components of belowground communities follow divergent responses to soil properties and land use intensification; however, it is unclear whether this extends across heterogeneous ecosystems. Here, a national-scale metabarcoding analysis of 436 locations across 7 different temperate ecosystems shows that belowground animal and microbial (bacteria, archaea, fungi, and protists) richness follow divergent trends, whereas β-diversity does not. Animal richness is governed by intensive land use and unaffected by soil properties, while microbial richness was driven by environmental properties across land uses. Our findings demonstrate that established divergent patterns of belowground microbial and animal diversity are consistent across heterogeneous land uses and are detectable using a standardised metabarcoding approach.

Tuc2009 is a P335-type member of the tailed-phage supergroupLactic acid bacteria are economically important bacteria used in the production of fermented foods such as cheeses, yogurts, and sausages. Tuc2009 is a 38,347-bp lysogenic member of the P335 type of the Siphoviridae supergroup of noncontractile-tailed bacteriophages (GenBank accession no. NC_002703) and was originally identified in Lactococcus lactis subsp. cremoris UC509, a strain used in Cheddar cheese production, following mitomycin C induction (2, 42).Muralytic enzymes or lysins degrade the peptidoglycan (PG) matrix and play essential roles for both phages and bacteria. "Autolysins" is the term used for lysins which are produced by bacteria and involved in cell division, while the term "endolysins" refers to lytic enzymes involved in phage release. Some bacteria also produce lysins which act as class III bacteriocins. Lysins fall into three categories, glycosidases, amidases, and endopeptidases, depending on the type of chemical bond they cleave within the PG. Glycosidases can be further subdivided into the muramidases, glucosaminidases, and transglycosylases (55). Progeny release for many double-stranded-DNA-tailed phages has been shown to employ a lysis system involving one or more holins in conjunction with an endolysin. The holins function by forming pores in the cytoplasmic membrane of the host, thereby abolishing membrane potential and allowing the endolysin to access the PG layer.Lysins exhibit a modular design (16). While a portion (usually the N-terminal part in the case of endolysins) encodes bond cleavage, a second segment is involved in substrate binding. This is believed to help the enzymatic efficiency and specificity of such muralytic enzymes by locating the active motif directly at the site of the substrate and causing endolysins to lyse only those bacteria possessing both the specifically recognized binding region and the target bond of the cleaving domain. It is this specificity of target recognition that could make lysins attractive therapeutic agents. Indeed, studies have demonstrated the usefulness of lysins by specifically lysing streptococci which had colonized mice (38). The lysin is thus said to demonstrate independently functioning domains, as shown for the choline-binding motif of the majority of lysins of Streptococcus pneumoniae and its phages (16) and the endolysin of Tuc2009 (50). Furthermore, the level of homology between these modules from endolysins and autolysins is supportive of the modular theory of phage evolution, as it indicates that the genes encoding such enzymes have arisen as a result of genomic exchange and rearrangement (16).While the cellular PG layer gives structural support to the bacterium, it also represents a formidable barrier across which the phage must transport its DNA during the infection process. Several proteins used by phages infecting gram-negative bacteria to perform this task of "hole punching" have been characterized (45). Phages T4, T7, PRD1, and 6, all of which infect gram-negative ho...