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September 22, 2019

Avian transcriptomics: opportunities and challenges

Recent developments in next-generation sequencing technologies have greatly facilitated the study of whole transcriptomes in model and non-model species. Studying the transcriptome and how it changes across a variety of biological conditions has had major implications for our understanding of how the genome is regulated in different contexts, and how to interpret adaptations and the phenotype of an organism. The aim of this review is to highlight the potential of these new technologies for the study of avian transcriptomics, and to summarise how transcriptomics has been applied in ornithology. A total of 81 peer-reviewed scientific articles that used transcriptomics to answer questions within a broad range of study areas in birds are used as examples throughout the review. We further provide a quick guide to highlight the most important points which need to be take into account when planning a transcriptomic study in birds, and discuss how researchers with little background in molecular biology can avoid potential pitfalls. Suggestions for further reading are supplied throughout. We also discuss possible future developments in the technology platforms used for ribonucleic acid sequencing. By summarising how these novel technologies can be used to answer questions that have long been asked by ornithologists, we hope to bridge the gap between traditional ornithology and genomics, and to stimulate more interdisciplinary research.

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September 22, 2019

Single-cell (meta-)genomics of a dimorphic Candidatus Thiomargarita nelsonii reveals genomic plasticity.

The genus Thiomargarita includes the world’s largest bacteria. But as uncultured organisms, their physiology, metabolism, and basis for their gigantism are not well understood. Thus, a genomics approach, applied to a single Candidatus Thiomargarita nelsonii cell was employed to explore the genetic potential of one of these enigmatic giant bacteria. The Thiomargarita cell was obtained from an assemblage of budding Ca. T. nelsonii attached to a provannid gastropod shell from Hydrate Ridge, a methane seep offshore of Oregon, USA. Here we present a manually curated genome of Bud S10 resulting from a hybrid assembly of long Pacific Biosciences and short Illumina sequencing reads. With respect to inorganic carbon fixation and sulfur oxidation pathways, the Ca. T. nelsonii Hydrate Ridge Bud S10 genome was similar to marine sister taxa within the family Beggiatoaceae. However, the Bud S10 genome contains genes suggestive of the genetic potential for lithotrophic growth on arsenite and perhaps hydrogen. The genome also revealed that Bud S10 likely respires nitrate via two pathways: a complete denitrification pathway and a dissimilatory nitrate reduction to ammonia pathway. Both pathways have been predicted, but not previously fully elucidated, in the genomes of other large, vacuolated, sulfur-oxidizing bacteria. Surprisingly, the genome also had a high number of unusual features for a bacterium to include the largest number of metacaspases and introns ever reported in a bacterium. Also present, are a large number of other mobile genetic elements, such as insertion sequence (IS) transposable elements and miniature inverted-repeat transposable elements (MITEs). In some cases, mobile genetic elements disrupted key genes in metabolic pathways. For example, a MITE interrupts hupL, which encodes the large subunit of the hydrogenase in hydrogen oxidation. Moreover, we detected a group I intron in one of the most critical genes in the sulfur oxidation pathway, dsrA. The dsrA group I intron also carried a MITE sequence that, like the hupL MITE family, occurs broadly across the genome. The presence of a high degree of mobile elements in genes central to Thiomargarita’s core metabolism has not been previously reported in free-living bacteria and suggests a highly mutable genome.

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September 22, 2019

A community-based culture collection for targeting novel plant growth-promoting bacteria from the sugarcane microbiome.

The soil-plant ecosystem harbors an immense microbial diversity that challenges investigative approaches to study traits underlying plant-microbe association. Studies solely based on culture-dependent techniques have overlooked most microbial diversity. Here we describe the concomitant use of culture-dependent and -independent techniques to target plant-beneficial microbial groups from the sugarcane microbiome. The community-based culture collection (CBC) approach was used to access microbes from roots and stalks. The CBC recovered 399 unique bacteria representing 15.9% of the rhizosphere core microbiome and 61.6-65.3% of the endophytic core microbiomes of stalks. By cross-referencing the CBC (culture-dependent) with the sugarcane microbiome profile (culture-independent), we designed a synthetic community comprised of naturally occurring highly abundant bacterial groups from roots and stalks, most of which has been poorly explored so far. We then used maize as a model to probe the abundance-based synthetic inoculant. We show that when inoculated in maize plants, members of the synthetic community efficiently colonize plant organs, displace the natural microbiota and dominate at 53.9% of the rhizosphere microbial abundance. As a result, inoculated plants increased biomass by 3.4-fold as compared to uninoculated plants. The results demonstrate that abundance-based synthetic inoculants can be successfully applied to recover beneficial plant microbes from plant microbiota.

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September 22, 2019

The state of play in higher eukaryote gene annotation.

A genome sequence is worthless if it cannot be deciphered; therefore, efforts to describe – or ‘annotate’ – genes began as soon as DNA sequences became available. Whereas early work focused on individual protein-coding genes, the modern genomic ocean is a complex maelstrom of alternative splicing, non-coding transcription and pseudogenes. Scientists – from clinicians to evolutionary biologists – need to navigate these waters, and this has led to the design of high-throughput, computationally driven annotation projects. The catalogues that are being produced are key resources for genome exploration, especially as they become integrated with expression, epigenomic and variation data sets. Their creation, however, remains challenging.

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September 22, 2019

Bacteroides dorei dominates gut microbiome prior to autoimmunity in Finnish children at high risk for type 1 diabetes.

The incidence of the autoimmune disease, type 1 diabetes (T1D), has increased dramatically over the last half century in many developed countries and is particularly high in Finland and other Nordic countries. Along with genetic predisposition, environmental factors are thought to play a critical role in this increase. As with other autoimmune diseases, the gut microbiome is thought to play a potential role in controlling progression to T1D in children with high genetic risk, but we know little about how the gut microbiome develops in children with high genetic risk for T1D. In this study, the early development of the gut microbiomes of 76 children at high genetic risk for T1D was determined using high-throughput 16S rRNA gene sequencing. Stool samples from children born in the same hospital in Turku, Finland were collected at monthly intervals beginning at 4-6 months after birth until 2.2 years of age. Of those 76 children, 29 seroconverted to T1D-related autoimmunity (cases) including 22 who later developed T1D, the remaining 47 subjects remained healthy (controls). While several significant compositional differences in low abundant species prior to seroconversion were found, one highly abundant group composed of two closely related species, Bacteroides dorei and Bacteroides vulgatus, was significantly higher in cases compared to controls prior to seroconversion. Metagenomic sequencing of samples high in the abundance of the B. dorei/vulgatus group before seroconversion, as well as longer 16S rRNA sequencing identified this group as Bacteroides dorei. The abundance of B. dorei peaked at 7.6 months in cases, over 8 months prior to the appearance of the first islet autoantibody, suggesting that early changes in the microbiome may be useful for predicting T1D autoimmunity in genetically susceptible infants. The cause of increased B. dorei abundance in cases is not known but its timing appears to coincide with the introduction of solid food.

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September 22, 2019

Analyses of intestinal microbiota: culture versus sequencing.

Analyzing human as well as animal microbiota composition has gained growing interest because structural components and metabolites of microorganisms fundamentally influence all aspects of host physiology. Originally dominated by culture-dependent methods for exploring these ecosystems, the development of molecular techniques such as high throughput sequencing has dramatically increased our knowledge. Because many studies of the microbiota are based on the bacterial 16S ribosomal RNA (rRNA) gene targets, they can, at least in principle, be compared to determine the role of the microbiome composition for developmental processes, host metabolism, and physiology as well as different diseases. In our review, we will summarize differences and pitfalls in current experimental protocols, including all steps from nucleic acid extraction to bioinformatical analysis which may produce variation that outweighs subtle biological differences. Future developments, such as integration of metabolomic, transcriptomic, and metagenomic data sets and standardization of the procedures, will be discussed. © The Author 2015. Published by Oxford University Press on behalf of the Institute for Laboratory Animal Research. All rights reserved. For permissions, please email: journals.permissions@oup.com.

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September 22, 2019

Ecological genomics of tropical trees: how local population size and allelic diversity of resistance genes relate to immune responses, cosusceptibility to pathogens, and negative density dependence

In tropical forests, rarer species show increased sensitivity to species-specific soil pathogens and more negative effects of conspecific density on seedling survival (NDD). These patterns suggest a connection between ecology and immunity, perhaps because small population size disproportionately reduces genetic diversity of hyperdiverse loci such as immunity genes. In an experiment examining seedling roots from six species in one tropical tree community, we found that smaller populations have reduced amino acid diversity in pathogen resistance (R) genes but not the transcriptome in general. Normalized R gene amino acid diversity varied with local abundance and prior measures of differences in sensitivity to conspecific soil and NDD. After exposure to live soil, species with lower R gene diversity had reduced defence gene induction, more cosusceptibility of maternal cohorts to colonization by potentially pathogenic fungi, reduced root growth arrest (an R gene-mediated response) and their root-associated fungi showed lower induction of self-defence (antioxidants). Local abundance was not related to the ability to induce immune responses when pathogen recognition was bypassed by application of salicylic acid, a phytohormone that activates defence responses downstream of R gene signalling. These initial results support the hypothesis that smaller local tree populations have reduced R gene diversity and recognition-dependent immune responses, along with greater cosusceptibility to species-specific pathogens that may facilitate disease transmission and NDD. Locally rare species may be less able to increase their equilibrium abundance without genetic boosts to defence via immigration of novel R gene alleles from a larger and more diverse regional population.

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September 22, 2019

Role of clinicogenomics in infectious disease diagnostics and public health microbiology.

Clinicogenomics is the exploitation of genome sequence data for diagnostic, therapeutic, and public health purposes. Central to this field is the high-throughput DNA sequencing of genomes and metagenomes. The role of clinicogenomics in infectious disease diagnostics and public health microbiology was the topic of discussion during a recent symposium (session 161) presented at the 115th general meeting of the American Society for Microbiology that was held in New Orleans, LA. What follows is a collection of the most salient and promising aspects from each presentation at the symposium. Copyright © 2016, American Society for Microbiology. All Rights Reserved.

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September 22, 2019

First insights into the nature and evolution of antisense transcription in nematodes.

The development of multicellular organisms is coordinated by various gene regulatory mechanisms that ensure correct spatio-temporal patterns of gene expression. Recently, the role of antisense transcription in gene regulation has moved into focus of research. To characterize genome-wide patterns of antisense transcription and to study their evolutionary conservation, we sequenced a strand-specific RNA-seq library of the nematode Pristionchus pacificus.We identified 1112 antisense configurations of which the largest group represents 465 antisense transcripts (ASTs) that are fully embedded in introns of their host genes. We find that most ASTs show homology to protein-coding genes and are overrepresented in proteomic data. Together with the finding, that expression levels of ASTs and host genes are uncorrelated, this indicates that most ASTs in P. pacificus do not represent non-coding RNAs and do not exhibit regulatory functions on their host genes. We studied the evolution of antisense gene pairs across 20 nematode genomes, showing that the majority of pairs is lineage-specific and even the highly conserved vps-4, ddx-27, and sel-2 loci show abundant structural changes including duplications, deletions, intron gains and loss of antisense transcription. In contrast, host genes in general, are remarkably conserved and encode exceptionally long introns leading to unusually large blocks of conserved synteny.Our study has shown that in P. pacificus antisense transcription as such does not define non-coding RNAs but is rather a feature of highly conserved genes with long introns. We hypothesize that the presence of regulatory elements imposes evolutionary constraint on the intron length, but simultaneously, their large size makes them a likely target for translocation of genomic elements including protein-coding genes that eventually end up as ASTs.

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September 22, 2019

Microsatellites from Fosterella christophii (Bromeliaceae) by de novo transcriptome sequencing on the Pacific Biosciences RS platform.

Microsatellite markers were developed in Fosterella christophii (Bromeliaceae) to investigate the genetic diversity and population structure within the F. micrantha group, comprising F. christophii, F. micrantha, and F. villosula.Full-length cDNAs were isolated from F. christophii and sequenced on a Pacific Biosciences RS platform. A total of 1590 high-quality consensus isoforms were assembled into 971 unigenes containing 421 perfect microsatellites. Thirty primer sets were designed, of which 13 revealed a high level of polymorphism in three populations of F. christophii, with four to nine alleles per locus. Each of these 13 loci cross-amplified in the closely related species F. micrantha and F. villosula, with one to six and one to 11 alleles per locus, respectively.The new markers are promising tools to study the population genetics of F. christophii and to discover species boundaries within the F. micrantha group.

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September 22, 2019

Isoform sequencing provides insight into natural genetic diversity in maize.

W64A, as a member of non-stiff stalk maize, has been used to develop current corn in plant breeding, and serving as one of broadest parent line for the commercial hybrid seed production (Huffman, 1984). The inbred had the characteristics of early flowering, average plant and ear height at its maturity, very strong roots and good stalks (Runge, 2004). In addition, W64A serves as an invaluable germplasm to study gene functions especially in the field of corn nutrition and endosperm texture given its nearly complete vitreousness and hardness (Figure 1a). However, little is known about the background of genetic and genomic information for W64A. With the advent of the revolutionary technology of PacBio long-read sequencing, we can simultaneously obtain a large amount of full-length cDNA up to 20 kb (An et al., 2018). This article is protected by copyright. All rights reserved.This article is protected by copyright. All rights reserved.

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September 22, 2019

High-resolution characterization of the human microbiome.

The human microbiome plays an important and increasingly recognized role in human health. Studies of the microbiome typically use targeted sequencing of the 16S rRNA gene, whole metagenome shotgun sequencing, or other meta-omic technologies to characterize the microbiome’s composition, activity, and dynamics. Processing, analyzing, and interpreting these data involve numerous computational tools that aim to filter, cluster, annotate, and quantify the obtained data and ultimately provide an accurate and interpretable profile of the microbiome’s taxonomy, functional capacity, and behavior. These tools, however, are often limited in resolution and accuracy and may fail to capture many biologically and clinically relevant microbiome features, such as strain-level variation or nuanced functional response to perturbation. Over the past few years, extensive efforts have been invested toward addressing these challenges and developing novel computational methods for accurate and high-resolution characterization of microbiome data. These methods aim to quantify strain-level composition and variation, detect and characterize rare microbiome species, link specific genes to individual taxa, and more accurately characterize the functional capacity and dynamics of the microbiome. These methods and the ability to produce detailed and precise microbiome information are clearly essential for informing microbiome-based personalized therapies. In this review, we survey these methods, highlighting the challenges each method sets out to address and briefly describing methodological approaches. Copyright © 2016 Elsevier Inc. All rights reserved.

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September 22, 2019

A quantitative SMRT cell sequencing method for ribosomal amplicons.

Advances in sequencing technologies continue to provide unprecedented opportunities to characterize microbial communities. For example, the Pacific Biosciences Single Molecule Real-Time (SMRT) platform has emerged as a unique approach harnessing DNA polymerase activity to sequence template molecules, enabling long reads at low costs. With the aim to simultaneously classify and enumerate in situ microbial populations, we developed a quantitative SMRT (qSMRT) approach that involves the addition of exogenous standards to quantify ribosomal amplicons derived from environmental samples. The V7-9 regions of 18S SSU rDNA were targeted and quantified from protistan community samples collected in the Ross Sea during the Austral summer of 2011. We used three standards of different length and optimized conditions to obtain accurate quantitative retrieval across the range of expected amplicon sizes, a necessary criterion for analyzing taxonomically diverse 18S rDNA molecules from natural environments. The ability to concurrently identify and quantify microorganisms in their natural environment makes qSMRT a powerful, rapid and cost-effective approach for defining ecosystem diversity and function. Copyright © 2017 Elsevier B.V. All rights reserved.

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September 22, 2019

Enigmatic Diphyllatea eukaryotes: culturing and targeted PacBio RS amplicon sequencing reveals a higher order taxonomic diversity and global distribution.

The class Diphyllatea belongs to a group of enigmatic unicellular eukaryotes that play a key role in reconstructing the morphological innovation and diversification of early eukaryotic evolution. Despite its evolutionary significance, very little is known about the phylogeny and species diversity of Diphyllatea. Only three species have described morphology, being taxonomically divided by flagella number, two or four, and cell size. Currently, one 18S rRNA Diphyllatea sequence is available, with environmental sequencing surveys reporting only a single partial sequence from a Diphyllatea-like organism. Accordingly, geographical distribution of Diphyllatea based on molecular data is limited, despite morphological data suggesting the class has a global distribution. We here present a first attempt to understand species distribution, diversity and higher order structure of Diphyllatea.We cultured 11 new strains, characterised these morphologically and amplified their rRNA for a combined 18S-28S rRNA phylogeny. We sampled environmental DNA from multiple sites and designed new Diphyllatea-specific PCR primers for long-read PacBio RSII technology. Near full-length 18S rRNA sequences from environmental DNA, in addition to supplementary Diphyllatea sequence data mined from public databases, resolved the phylogeny into three deeply branching and distinct clades (Diphy I – III). Of these, the Diphy III clade is entirely novel, and in congruence with Diphy II, composed of species morphologically consistent with the earlier described Collodictyon triciliatum. The phylogenetic split between the Diphy I and Diphy II?+?III clades corresponds with a morphological division of Diphyllatea into bi- and quadriflagellate cell forms.This altered flagella composition must have occurred early in the diversification of Diphyllatea and may represent one of the earliest known morphological transitions among eukaryotes. Further, the substantial increase in molecular data presented here confirms Diphyllatea has a global distribution, seemingly restricted to freshwater habitats. Altogether, the results reveal the advantage of combining a group-specific PCR approach and long-read high-throughput amplicon sequencing in surveying enigmatic eukaryote lineages. Lastly, our study shows the capacity of PacBio RS when targeting a protist class for increasing phylogenetic resolution.

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September 22, 2019

Metagenomic approaches to assess bacteriophages in various environmental niches.

Bacteriophages are ubiquitous and numerous parasites of bacteria and play a critical evolutionary role in virtually every ecosystem, yet our understanding of the extent of the diversity and role of phages remains inadequate for many ecological niches, particularly in cases in which the host is unculturable. During the past 15 years, the emergence of the field of viral metagenomics has drastically enhanced our ability to analyse the so-called viral ‘dark matter’ of the biosphere. Here, we review the evolution of viral metagenomic methodologies, as well as providing an overview of some of the most significant applications and findings in this field of research.

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