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September 22, 2019

The bacterial microbiome of Dermacentor andersoni ticks influences pathogen susceptibility.

Ticks are of medical importance owing to their ability to transmit pathogens to humans and animals. The Rocky Mountain wood tick, Dermacentor andersoni, is a vector of a number of pathogens, including Anaplasma marginale, which is the most widespread tick-borne pathogen of livestock. Although ticks host pathogenic bacteria, they also harbor bacterial endosymbionts that have a role in tick physiology, survival, as well as pathogen acquisition and transmission. The goal of this study was to characterize the bacterial microbiome and examine the impact of microbiome disruption on pathogen susceptibility. The bacterial microbiome of two populations of D. andersoni with historically different susceptibilities to A. marginale was characterized. In this study, the microbiome was disrupted and then ticks were exposed to A. marginale or Francisella novicida to determine whether the microbiome correlated with pathogen susceptibility. Our study showed that an increase in proportion and quantity of Rickettsia bellii in the microbiome was negatively correlated to A. marginale levels in ticks. Furthermore, a decrease in Francisella endosymbionts was associated with lower F. novicida infection levels, demonstrating a positive pathogen-endosymbiont relationship. We demonstrate that endosymbionts and pathogens have varying interactions, and suggest that microbiome manipulation may provide a possible method for biocontrol by decreasing pathogen susceptibility of ticks.

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September 22, 2019

Dynamic transcriptome profiling dataset of vaccinia virus obtained from long-read sequencing techniques.

Poxviruses are large DNA viruses that infect humans and animals. Vaccinia virus (VACV) has been applied as a live vaccine for immunization against smallpox, which was eradicated by 1980 as a result of worldwide vaccination. VACV is the prototype of poxviruses in the investigation of the molecular pathogenesis of the virus. Short-read sequencing methods have revolutionized transcriptomics; however, they are not efficient in distinguishing between the RNA isoforms and transcript overlaps. Long-read sequencing (LRS) is much better suited to solve these problems and also allow direct RNA sequencing. Despite the scientific relevance of VACV, no LRS data have been generated for the viral transcriptome to date.For the deep characterization of the VACV RNA profile, various LRS platforms and library preparation approaches were applied. The raw reads were mapped to the VACV reference genome and also to the host (Chlorocebus sabaeus) genome. In this study, we applied the Pacific Biosciences RSII and Sequel platforms, which altogether resulted in 937,531 mapped reads of inserts (1.42 Gb), while we obtained 2,160,348 aligned reads (1.75 Gb) from the different library preparation methods using the MinION device from Oxford Nanopore Technologies.By applying cutting-edge technologies, we were able to generate a large dataset that can serve as a valuable resource for the investigation of the dynamic VACV transcriptome, the virus-host interactions, and RNA base modifications. These data can provide useful information for novel gene annotations in the VACV genome. Our dataset can also be used to analyze the currently available LRS platforms, library preparation methods, and bioinformatics pipelines.

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September 22, 2019

Limited effects of variable-retention harvesting on fungal communities decomposing fine roots in coastal temperate rainforests.

Fine root litter is the principal source of carbon stored in forest soils and a dominant source of carbon for fungal decomposers. Differences in decomposer capacity between fungal species may be important determinants of fine-root decomposition rates. Variable-retention harvesting (VRH) provides refuge for ectomycorrhizal fungi, but its influence on fine-root decomposers is unknown, as are the effects of functional shifts in these fungal communities on carbon cycling. We compared fungal communities decomposing fine roots (in litter bags) under VRH, clear-cut, and uncut stands at two sites (6 and 13 years postharvest) and two decay stages (43 days and 1 year after burial) in Douglas fir forests in coastal British Columbia, Canada. Fungal species and guilds were identified from decomposed fine roots using high-throughput sequencing. Variable retention had short-term effects on ß-diversity; harvest treatment modified the fungal community composition at the 6-year-postharvest site, but not at the 13-year-postharvest site. Ericoid and ectomycorrhizal guilds were not more abundant under VRH, but stand age significantly structured species composition. Guild composition varied by decay stage, with ruderal species later replaced by saprotrophs and ectomycorrhizae. Ectomycorrhizal abundance on decomposing fine roots may partially explain why fine roots typically decompose more slowly than surface litter. Our results indicate that stand age structures fine-root decomposers but that decay stage is more important in structuring the fungal community than shifts caused by harvesting. The rapid postharvest recovery of fungal communities decomposing fine roots suggests resiliency within this community, at least in these young regenerating stands in coastal British Columbia.IMPORTANCE Globally, fine roots are a dominant source of carbon in forest soils, yet the fungi that decompose this material and that drive the sequestration or respiration of this carbon remain largely uncharacterized. Fungi vary in their capacity to decompose plant litter, suggesting that fungal community composition is an important determinant of decomposition rates. Variable-retention harvesting is a forestry practice that modifies fungal communities by providing refuge for ectomycorrhizal fungi. We evaluated the effects of variable retention and clear-cut harvesting on fungal communities decomposing fine roots at two sites (6 and 13 years postharvest), at two decay stages (43 days and 1 year), and in uncut stands in temperate rainforests. Harvesting impacts on fungal community composition were detected only after 6 years after harvest. We suggest that fungal community composition may be an important factor that reduces fine-root decomposition rates relative to those of above-ground plant litter, which has important consequences for forest carbon cycling. Copyright © 2018 American Society for Microbiology.

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September 22, 2019

Separation and parallel sequencing of the genomes and transcriptomes of single cells using G&T-seq.

Parallel sequencing of a single cell’s genome and transcriptome provides a powerful tool for dissecting genetic variation and its relationship with gene expression. Here we present a detailed protocol for G&T-seq, a method for separation and parallel sequencing of genomic DNA and full-length polyA(+) mRNA from single cells. We provide step-by-step instructions for the isolation and lysis of single cells; the physical separation of polyA(+) mRNA from genomic DNA using a modified oligo-dT bead capture and the respective whole-transcriptome and whole-genome amplifications; and library preparation and sequence analyses of these amplification products. The method allows the detection of thousands of transcripts in parallel with the genetic variants captured by the DNA-seq data from the same single cell. G&T-seq differs from other currently available methods for parallel DNA and RNA sequencing from single cells, as it involves physical separation of the DNA and RNA and does not require bespoke microfluidics platforms. The process can be implemented manually or through automation. When performed manually, paired genome and transcriptome sequencing libraries from eight single cells can be produced in ~3 d by researchers experienced in molecular laboratory work. For users with experience in the programming and operation of liquid-handling robots, paired DNA and RNA libraries from 96 single cells can be produced in the same time frame. Sequence analysis and integration of single-cell G&T-seq DNA and RNA data requires a high level of bioinformatics expertise and familiarity with a wide range of informatics tools.

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September 22, 2019

Evaluating the mobility potential of antibiotic resistance genes in environmental resistomes without metagenomics.

Antibiotic resistance genes are ubiquitous in the environment. However, only a fraction of them are mobile and able to spread to pathogenic bacteria. Until now, studying the mobility of antibiotic resistance genes in environmental resistomes has been challenging due to inadequate sensitivity and difficulties in contig assembly of metagenome based methods. We developed a new cost and labor efficient method based on Inverse PCR and long read sequencing for studying mobility potential of environmental resistance genes. We applied Inverse PCR on sediment samples and identified 79 different MGE clusters associated with the studied resistance genes, including novel mobile genetic elements, co-selected resistance genes and a new putative antibiotic resistance gene. The results show that the method can be used in antibiotic resistance early warning systems. In comparison to metagenomics, Inverse PCR was markedly more sensitive and provided more data on resistance gene mobility and co-selected resistances.

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September 22, 2019

Differential expression analysis of olfactory genes based on a combination of sequencing platforms and behavioral investigations in Aphidius gifuensis.

Aphidius gifuensis Ashmead is a dominant endoparasitoid of aphids, such as Myzus persicae and Sitobion avenae, and plays an important role in controlling aphids in various habitats, including tobacco plants and wheat in China. A. gifuensis has been successfully applied for the biological control of aphids, especially M. persicae, in green houses and fields in China. The corresponding parasites, as well as its mate-searching behaviors, are subjects of considerable interest. Previous A. gifuensis transcriptome studies have relied on short-read next-generation sequencing (NGS), and the vast majority of the resulting isotigs do not represent full-length cDNA. Here, we employed a combination of NGS and single-molecule real-time (SMRT) sequencing of virgin females (VFs), mated females (MFs), virgin males (VMs), and mated males (MMs) to comprehensively study the A. gifuensis transcriptome. Behavioral responses to the aphid alarm pheromone (E-ß-farnesene, EBF) as well as to A. gifuensis of the opposite sex were also studied. VMs were found to be attracted by female wasps and MFs were repelled by male wasps, whereas MMs and VFs did not respond to the opposite sex. In addition, VFs, MFs, and MMs were attracted by EBF, while VMs did not respond. According to these results, we performed a personalized differential gene expression analysis of olfactory gene sets (66 odorant receptors, 25 inotropic receptors, 16 odorant-binding proteins, and 12 chemosensory proteins) in virgin and mated A. gifuensis of both sexes, and identified 13 candidate genes whose expression levels were highly consistent with behavioral test results, suggesting potential functions for these genes in pheromone perception.

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September 22, 2019

Influenza virus infection causes global RNAPII termination defects.

Viral infection perturbs host cells and can be used to uncover regulatory mechanisms controlling cellular responses and susceptibility to infections. Using cell biological, biochemical, and genetic tools, we reveal that influenza A virus (IAV) infection induces global transcriptional defects at the 3′ ends of active host genes and RNA polymerase II (RNAPII) run-through into extragenic regions. Deregulated RNAPII leads to expression of aberrant RNAs (3′ extensions and host-gene fusions) that ultimately cause global transcriptional downregulation of physiological transcripts, an effect influencing antiviral response and virulence. This phenomenon occurs with multiple strains of IAV, is dependent on influenza NS1 protein, and can be modulated by SUMOylation of an intrinsically disordered region (IDR) of NS1 expressed by the 1918 pandemic IAV strain. Our data identify a strategy used by IAV to suppress host gene expression and indicate that polymorphisms in IDRs of viral proteins can affect the outcome of an infection.

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September 22, 2019

Contrasting distribution patterns between aquatic and terrestrial Phytophthora species along a climatic gradient are linked to functional traits.

Diversity of microbial organisms is linked to global climatic gradients. The genus Phytophthora includes both aquatic and terrestrial plant pathogenic species that display a large variation of functional traits. The extent to which the physical environment (water or soil) modulates the interaction of microorganisms with climate is unknown. Here, we explored the main environmental drivers of diversity and functional trait composition of Phytophthora communities. Communities were obtained by a novel metabarcoding setup based on PacBio sequencing of river filtrates in 96 river sites along a geographical gradient. Species were classified as terrestrial or aquatic based on their phylogenetic clade. Overall, terrestrial and aquatic species showed contrasting patterns of diversity. For terrestrial species, precipitation was a stronger driver than temperature, and diversity and functional diversity decreased with decreasing temperature and precipitation. In cold and dry areas, the dominant species formed resistant structures and had a low optimum temperature. By contrast, for aquatic species, temperature and water chemistry were the strongest drivers, and diversity increased with decreasing temperature and precipitation. Within the same area, environmental filtering affected terrestrial species more strongly than aquatic species (20% versus 3% of the studied communities, respectively). Our results highlight the importance of functional traits and the physical environment in which microorganisms develop their life cycle when predicting their distribution under changing climatic conditions. Temperature and rainfall may be buffered differently by water and soil, and thus pose contrasting constrains to microbial assemblies.

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September 22, 2019

Full-length transcriptome sequences and splice variants obtained by a combination of sequencing platforms applied to different root tissues of Salvia miltiorrhiza and tanshinone biosynthesis.

Danshen, Salvia miltiorrhiza Bunge, is one of the most widely used herbs in traditional Chinese medicine, wherein its rhizome/roots are particularly valued. The corresponding bioactive components include the tanshinone diterpenoids, the biosynthesis of which is a subject of considerable interest. Previous investigations of the S. miltiorrhiza transcriptome have relied on short-read next-generation sequencing (NGS) technology, and the vast majority of the resulting isotigs do not represent full-length cDNA sequences. Moreover, these efforts have been targeted at either whole plants or hairy root cultures. Here, we demonstrate that the tanshinone pigments are produced and accumulate in the root periderm, and apply a combination of NGS and single-molecule real-time (SMRT) sequencing to various root tissues, particularly including the periderm, to provide a more complete view of the S. miltiorrhiza transcriptome, with further insight into tanshinone biosynthesis as well. In addition, the use of SMRT long-read sequencing offered the ability to examine alternative splicing, which was found to occur in approximately 40% of the detected gene loci, including several involved in isoprenoid/terpenoid metabolism.© 2015 The Authors The Plant Journal © 2015 John Wiley & Sons Ltd.

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September 22, 2019

Gene activity in primary T cells infected with HIV89.6: intron retention and induction of genomic repeats.

HIV infection has been reported to alter cellular gene activity, but published studies have commonly assayed transformed cell lines and lab-adapted HIV strains, yielding inconsistent results. Here we carried out a deep RNA-Seq analysis of primary human T cells infected with the low passage HIV isolate HIV89.6.Seventeen percent of cellular genes showed altered activity 48 h after infection. In a meta-analysis including four other studies, our data differed from studies of HIV infection in cell lines but showed more parallels with infections of primary cells. We found a global trend toward retention of introns after infection, suggestive of a novel cellular response to infection. HIV89.6 infection was also associated with activation of several human endogenous retroviruses (HERVs) and retrotransposons, of interest as possible novel antigens that could serve as vaccine targets. The most highly activated group of HERVs was a subset of the ERV-9. Analysis showed that activation was associated with a particular variant of ERV-9 long terminal repeats that contains an indel near the U3-R border. These data also allowed quantification of >70 splice forms of the HIV89.6 RNA and specified the main types of chimeric HIV89.6-host RNAs. Comparison to over 100,000 integration site sequences from the same infected cell populations allowed quantification of authentic versus artifactual chimeric reads, showing that 5′ read-in, splicing out of HIV89.6 from the D4 donor and 3′ read-through were the most common HIV89.6-host cell chimeric RNA forms.Analysis of RNA abundance after infection of primary T cells with the low passage HIV89.6 isolate disclosed multiple novel features of HIV-host interactions, notably intron retention and induction of transcription of retrotransposons and endogenous retroviruses.

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September 22, 2019

PacBio sequencing of gene families – a case study with wheat gluten genes.

Amino acids in wheat (Triticum aestivum) seeds mainly accumulate in storage proteins called gliadins and glutenins. Gliadins contain a/ß-, ?- and ?-types whereas glutenins contain HMW- and LMW-types. Known gliadin and glutenin sequences were largely determined through cloning and sequencing by capillary electrophoresis. This time-consuming process prevents us to intensively study the variation of each orthologous gene copy among cultivars. The throughput and sequencing length of Pacific Bioscience RS (PacBio) single molecule sequencing platform make it feasible to construct contiguous and non-chimeric RNA sequences. We assembled 424 wheat storage protein transcripts from ten wheat cultivars by using just one single-molecule-real-time cell. The protein genes from wheat cultivar Chinese Spring are comparable to known sequences from NCBI. We demonstrated real-time sequencing of gene families with high-throughput and low-cost. This method can be applied to studies of gene amplification and copy number variation among species and cultivars. © 2013 Elsevier B.V. All rights reserved.

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September 22, 2019

Exploiting single-molecule transcript sequencing for eukaryotic gene prediction.

We develop a method to predict and validate gene models using PacBio single-molecule, real-time (SMRT) cDNA reads. Ninety-eight percent of full-insert SMRT reads span complete open reading frames. Gene model validation using SMRT reads is developed as automated process. Optimized training and prediction settings and mRNA-seq noise reduction of assisting Illumina reads results in increased gene prediction sensitivity and precision. Additionally, we present an improved gene set for sugar beet (Beta vulgaris) and the first genome-wide gene set for spinach (Spinacia oleracea). The workflow and guidelines are a valuable resource to obtain comprehensive gene sets for newly sequenced genomes of non-model eukaryotes.

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September 22, 2019

Single-Molecule Long-Read Sequencing of Zanthoxylum bungeanum Maxim. Transcriptome: Identification of Aroma-Related Genes

Zanthoxylum bungeanum Maxim. is an economically important tree species that is resistant to drought and infertility, and has potential medicinal and edible value. However, comprehensive genomic data are not yet available for this species, limiting its potential utility for medicinal use, breeding programs, and cultivation. Transcriptome sequencing provides an effective approach to remedying this shortcoming. Herein, single-molecule long-read sequencing and next-generation sequencingapproacheswereusedinparalleltoobtaintranscriptisoformstructureandgenefunctional informationinZ.bungeanum. Intotal, 282,101readsofinserts(ROIs)wereidentified, including134,074 full-length non-chimeric reads, among which 65,711 open reading frames (ORFs), 50,135 simple sequence repeats (SSRs), and 1492 long non-coding RNAs (lncRNAs) were detected. Functional annotation revealed metabolic pathways related to aroma components and color characteristics in Z. bungeanum. Unexpectedly, 30 transcripts were annotated as genes involved in regulating the pathogenesis of breast and colorectal cancers. This work provides a comprehensive transcriptome resource for Z. bungeanum, and lays a foundation for the further investigation and utilization of Zanthoxylum resources.

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September 22, 2019

The habu genome reveals accelerated evolution of venom protein genes.

Evolution of novel traits is a challenging subject in biological research. Several snake lineages developed elaborate venom systems to deliver complex protein mixtures for prey capture. To understand mechanisms involved in snake venom evolution, we decoded here the ~1.4-Gb genome of a habu, Protobothrops flavoviridis. We identified 60 snake venom protein genes (SV) and 224 non-venom paralogs (NV), belonging to 18 gene families. Molecular phylogeny reveals early divergence of SV and NV genes, suggesting that one of the four copies generated through two rounds of whole-genome duplication was modified for use as a toxin. Among them, both SV and NV genes in four major components were extensively duplicated after their diversification, but accelerated evolution is evident exclusively in the SV genes. Both venom-related SV and NV genes are significantly enriched in microchromosomes. The present study thus provides a genetic background for evolution of snake venom composition.

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September 22, 2019

Isoform sequencing and state-of-art applications for unravelling complexity of plant transcriptomes

Single-molecule real-time (SMRT) sequencing developed by PacBio, also called third-generation sequencing (TGS), offers longer reads than the second-generation sequencing (SGS). Given its ability to obtain full-length transcripts without assembly, isoform sequencing (Iso-Seq) of transcriptomes by PacBio is advantageous for genome annotation, identification of novel genes and isoforms, as well as the discovery of long non-coding RNA (lncRNA). In addition, Iso-Seq gives access to the direct detection of alternative splicing, alternative polyadenylation (APA), gene fusion, and DNA modifications. Such applications of Iso-Seq facilitate the understanding of gene structure, post-transcriptional regulatory networks, and subsequently proteomic diversity. In this review, we summarize its applications in plant transcriptome study, specifically pointing out challenges associated with each step in the experimental design and highlight the development of bioinformatic pipelines. We aim to provide the community with an integrative overview and a comprehensive guidance to Iso-Seq, and thus to promote its applications in plant research.

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