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September 22, 2019

Exploring the genome and transcriptome of the cave nectar bat Eonycteris spelaea with PacBio long-read sequencing.

In the past two decades, bats have emerged as an important model system to study host-pathogen interactions. More recently, it has been shown that bats may also serve as a new and excellent model to study aging, inflammation, and cancer, among other important biological processes. The cave nectar bat or lesser dawn bat (Eonycteris spelaea) is known to be a reservoir for several viruses and intracellular bacteria. It is widely distributed throughout the tropics and subtropics from India to Southeast Asia and pollinates several plant species, including the culturally and economically important durian in the region. Here, we report the whole-genome and transcriptome sequencing, followed by subsequent de novo assembly, of the E. spelaea genome solely using the Pacific Biosciences (PacBio) long-read sequencing platform.The newly assembled E. spelaea genome is 1.97 Gb in length and consists of 4,470 sequences with a contig N50 of 8.0 Mb. Identified repeat elements covered 34.65% of the genome, and 20,640 unique protein-coding genes with 39,526 transcripts were annotated.We demonstrated that the PacBio long-read sequencing platform alone is sufficient to generate a comprehensive de novo assembled genome and transcriptome of an important bat species. These results will provide useful insights and act as a resource to expand our understanding of bat evolution, ecology, physiology, immunology, viral infection, and transmission dynamics.

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September 22, 2019

Clinical PathoScope: rapid alignment and filtration for accurate pathogen identification in clinical samples using unassembled sequencing data.

The use of sequencing technologies to investigate the microbiome of a sample can positively impact patient healthcare by providing therapeutic targets for personalized disease treatment. However, these samples contain genomic sequences from various sources that complicate the identification of pathogens.Here we present Clinical PathoScope, a pipeline to rapidly and accurately remove host contamination, isolate microbial reads, and identify potential disease-causing pathogens. We have accomplished three essential tasks in the development of Clinical PathoScope. First, we developed an optimized framework for pathogen identification using a computational subtraction methodology in concordance with read trimming and ambiguous read reassignment. Second, we have demonstrated the ability of our approach to identify multiple pathogens in a single clinical sample, accurately identify pathogens at the subspecies level, and determine the nearest phylogenetic neighbor of novel or highly mutated pathogens using real clinical sequencing data. Finally, we have shown that Clinical PathoScope outperforms previously published pathogen identification methods with regard to computational speed, sensitivity, and specificity.Clinical PathoScope is the only pathogen identification method currently available that can identify multiple pathogens from mixed samples and distinguish between very closely related species and strains in samples with very few reads per pathogen. Furthermore, Clinical PathoScope does not rely on genome assembly and thus can more rapidly complete the analysis of a clinical sample when compared with current assembly-based methods. Clinical PathoScope is freely available at: http://sourceforge.net/projects/pathoscope/.

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September 22, 2019

Evolution of selective-sequencing approaches for virus discovery and virome analysis.

Recent advances in sequencing technologies have transformed the field of virus discovery and virome analysis. Once mostly confined to the traditional Sanger sequencing based individual virus discovery, is now entirely replaced by high throughput sequencing (HTS) based virus metagenomics that can be used to characterize the nature and composition of entire viromes. To better harness the potential of HTS for the study of viromes, sample preparation methodologies use different approaches to exclude amplification of non-viral components that can overshadow low-titer viruses. These virus-sequence enrichment approaches mostly focus on the sample preparation methods, like enzymatic digestion of non-viral nucleic acids and size exclusion of non-viral constituents by column filtration, ultrafiltration or density gradient centrifugation. However, recently a new approach of virus-sequence enrichment called virome-capture sequencing, focused on the amplification or HTS library preparation stage, was developed to increase the ability of virome characterization. This new approach has the potential to further transform the field of virus discovery and virome analysis, but its technical complexity and sequence-dependence warrants further improvements. In this review we discuss the different methods, their applications and evolution, for selective sequencing based virome analysis and also propose refinements needed to harness the full potential of HTS for virome analysis. Copyright © 2017 Elsevier B.V. All rights reserved.

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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

Computational identification of novel genes: current and future perspectives.

While it has long been thought that all genomic novelties are derived from the existing material, many genes lacking homology to known genes were found in recent genome projects. Some of these novel genes were proposed to have evolved de novo, ie, out of noncoding sequences, whereas some have been shown to follow a duplication and divergence process. Their discovery called for an extension of the historical hypotheses about gene origination. Besides the theoretical breakthrough, increasing evidence accumulated that novel genes play important roles in evolutionary processes, including adaptation and speciation events. Different techniques are available to identify genes and classify them as novel. Their classification as novel is usually based on their similarity to known genes, or lack thereof, detected by comparative genomics or against databases. Computational approaches are further prime methods that can be based on existing models or leveraging biological evidences from experiments. Identification of novel genes remains however a challenging task. With the constant software and technologies updates, no gold standard, and no available benchmark, evaluation and characterization of genomic novelty is a vibrant field. In this review, the classical and state-of-the-art tools for gene prediction are introduced. The current methods for novel gene detection are presented; the methodological strategies and their limits are discussed along with perspective approaches for further studies.

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September 22, 2019

Candidatus Dactylopiibacterium carminicum, a nitrogen-fixing symbiont of Dactylopius cochineal insects (Hemiptera: Coccoidea: Dactylopiidae)

The domesticated carmine cochineal Dactylopius coccus (scale insect) has commercial value and has been used for more than 500?years for natural red pigment production. Besides the domesticated cochineal, other wild Dactylopius species such as Dactylopius opuntiae are found in the Americas, all feeding on nutrient poor sap from native cacti. To compensate nutritional deficiencies, many insects harbor symbiotic bacteria which provide essential amino acids or vitamins to their hosts. Here, we characterized a symbiont from the carmine cochineal insects, Candidatus Dactylopiibacterium carminicum (betaproteobacterium, Rhodocyclaceae family) and found it in D. coccus and in D. opuntiae ovaries by fluorescent in situ hybridization, suggesting maternal inheritance. Bacterial genomes recovered from metagenomic data derived from whole insects or tissues both from D. coccus and from D. opuntiae were around 3.6?Mb in size. Phylogenomics showed that dactylopiibacteria constituted a closely related clade neighbor to nitrogen fixing bacteria from soil or from various plants including rice and other grass endophytes. Metabolic capabilities were inferred from genomic analyses, showing a complete operon for nitrogen fixation, biosynthesis of amino acids and vitamins and putative traits of anaerobic or microoxic metabolism as well as genes for plant interaction. Dactylopiibacterium nif gene expression and acetylene reduction activity detecting nitrogen fixation were evidenced in D. coccus hemolymph and ovaries, in congruence with the endosymbiont fluorescent in situ hybridization location. Dactylopiibacterium symbionts may compensate for the nitrogen deficiency in the cochineal diet. In addition, this symbiont may provide essential amino acids, recycle uric acid, and increase the cochineal life span.

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September 22, 2019

wtf genes are prolific dual poison-antidote meiotic drivers.

Meiotic drivers are selfish genes that bias their transmission into gametes, defying Mendelian inheritance. Despite the significant impact of these genomic parasites on evolution and infertility, few meiotic drive loci have been identified or mechanistically characterized. Here, we demonstrate a complex landscape of meiotic drive genes on chromosome 3 of the fission yeasts Schizosaccharomyces kambucha and S. pombe. We identify S. kambucha wtf4 as one of these genes that acts to kill gametes (known as spores in yeast) that do not inherit the gene from heterozygotes. wtf4 utilizes dual, overlapping transcripts to encode both a gamete-killing poison and an antidote to the poison. To enact drive, all gametes are poisoned, whereas only those that inherit wtf4 are rescued by the antidote. Our work suggests that the wtf multigene family proliferated due to meiotic drive and highlights the power of selfish genes to shape genomes, even while imposing tremendous costs to fertility.

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September 22, 2019

Rodent papillomaviruses.

Preclinical infection model systems are extremely valuable tools to aid in our understanding of Human Papillomavirus (HPV) biology, disease progression, prevention, and treatments. In this context, rodent papillomaviruses and their respective infection models are useful tools but remain underutilized resources in the field of papillomavirus biology. Two rodent papillomaviruses, MnPV1, which infects the Mastomys species of multimammate rats, and MmuPV1, which infects laboratory mice, are currently the most studied rodent PVs. Both of these viruses cause malignancy in the skin and can provide attractive infection models to study the lesser understood cutaneous papillomaviruses that have been frequently associated with HPV-related skin cancers. Of these, MmuPV1 is the first reported rodent papillomavirus that can naturally infect the laboratory strain of mice. MmuPV1 is an attractive model virus to study papillomavirus pathogenesis because of the ubiquitous availability of lab mice and the fact that this mouse species is genetically modifiable. In this review, we have summarized the knowledge we have gained about PV biology from the study of rodent papillomaviruses and point out the remaining gaps that can provide new research opportunities.

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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

Using PacBio long-read high-throughput microbial gene amplicon sequencing to evaluate infant formula safety.

Infant formula (IF) requires a strict microbiological standard because of the high vulnerability of infants to foodborne diseases. The current study used the PacBio single molecule real-time (SMRT) sequencing platform to generate full-length 16S rRNA-based bacterial microbiota profiles of thirty Chinese domestic and imported IF samples. A total of 600 species were identified, dominated by Streptococcus thermophilus, Lactococcus lactis and Lactococcus piscium. Distinctive bacterial profiles were observed between the two sample groups, as confirmed with both principal coordinate analysis and multivariate analysis of variance. Moreover, the product whey protein nitrogen index (WPNI), representing the degree of preheating, negatively correlated with the relative abundances of the Bacillus genus. Our study has demonstrated the application of the PacBio SMRT sequencing platform in assessing the bacterial contamination of IF products, which is of interest to the dairy industry for effective monitoring of microbial quality and safety during production.

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September 22, 2019

Distinguishing highly similar gene isoforms with a clustering-based bioinformatics analysis of PacBio single-molecule long reads.

Gene isoforms are commonly found in both prokaryotes and eukaryotes. Since each isoform may perform a specific function in response to changing environmental conditions, studying the dynamics of gene isoforms is important in understanding biological processes and disease conditions. However, genome-wide identification of gene isoforms is technically challenging due to the high degree of sequence identity among isoforms. Traditional targeted sequencing approach, involving Sanger sequencing of plasmid-cloned PCR products, has low throughput and is very tedious and time-consuming. Next-generation sequencing technologies such as Illumina and 454 achieve high throughput but their short read lengths are a critical barrier to accurate assembly of highly similar gene isoforms, and may result in ambiguities and false joining during sequence assembly. More recently, the third generation sequencer represented by the PacBio platform offers sufficient throughput and long reads covering the full length of typical genes, thus providing a potential to reliably profile gene isoforms. However, the PacBio long reads are error-prone and cannot be effectively analyzed by traditional assembly programs.We present a clustering-based analysis pipeline integrated with PacBio sequencing data for profiling highly similar gene isoforms. This approach was first evaluated in comparison to de novo assembly of 454 reads using a benchmark admixture containing 10 known, cloned msg genes encoding the major surface glycoprotein of Pneumocystis jirovecii. All 10 msg isoforms were successfully reconstructed with the expected length (~1.5 kb) and correct sequence by the new approach, while 454 reads could not be correctly assembled using various assembly programs. When using an additional benchmark admixture containing 22 known P. jirovecii msg isoforms, this approach accurately reconstructed all but 4 these isoforms in their full-length (~3 kb); these 4 isoforms were present in low concentrations in the admixture. Finally, when applied to the original clinical sample from which the 22 known msg isoforms were cloned, this approach successfully identified not only all known isoforms accurately (~3 kb each) but also 48 novel isoforms.PacBio sequencing integrated with the clustering-based analysis pipeline achieves high-throughput and high-resolution discrimination of highly similar sequences, and can serve as a new approach for genome-wide characterization of gene isoforms and other highly repetitive sequences.

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September 22, 2019

A high-quality annotated transcriptome of swine peripheral blood.

High throughput gene expression profiling assays of peripheral blood are widely used in biomedicine, as well as in animal genetics and physiology research. Accurate, comprehensive, and precise interpretation of such high throughput assays relies on well-characterized reference genomes and/or transcriptomes. However, neither the reference genome nor the peripheral blood transcriptome of the pig have been sufficiently assembled and annotated to support such profiling assays in this emerging biomedical model organism. We aimed to assemble published and novel RNA-seq data to provide a comprehensive, well-annotated blood transcriptome for pigs by integrating a de novo assembly with a genome-guided assembly.A de novo and a genome-guided transcriptome of porcine whole peripheral blood was assembled with ~162 million pairs of paired-end and ~183 million single-end, trimmed and normalized Illumina RNA-seq reads (~6 billion initial reads from 146 RNA-seq libraries) from five independent studies by using the Trinity and Cufflinks software, respectively. We then removed putative transcripts (PTs) of low confidence from both assemblies and merged the remaining PTs into an integrated transcriptome consisting of 132,928 PTs, with 126,225 (~95%) PTs from the de novo assembly and more than 91% of PTs spliced. In the integrated transcriptome, ~90% and 63% of PTs had significant sequence similarity to sequences in the NCBI NT and NR databases, respectively; 68,754 (~52%) PTs were annotated with 15,965 unique gene ontology (GO) terms; and 7618 PTs annotated with Enzyme Commission codes were assigned to 134 pathways curated by the Kyoto Encyclopedia of Genes and Genomes (KEGG). Full exon-intron junctions of 17,528 PTs were validated by PacBio IsoSeq full-length cDNA reads from 3 other porcine tissues, NCBI pig RefSeq mRNAs and transcripts from Ensembl Sscrofa10.2 annotation. Completeness of the 5′ termini of 37,569 PTs was validated by public cap analysis of gene expression (CAGE) data. By comparison to the Ensembl transcripts, we found that (1) the deduced precursors of 54,402 PTs shared at least one intron or exon with those of 18,437 Ensembl transcripts; (2) 12,262 PTs had both longer 5′ and 3′ termini than their maximally overlapping Ensembl transcripts; and (3) 41,838 spliced PTs were totally missing from the Sscrofa10.2 annotation. Similar results were obtained when the PTs were compared to the pig NCBI RefSeq mRNA collection.We built, validated and annotated a comprehensive porcine blood transcriptome with significant improvement over the annotation of Ensembl Sscrofa10.2 and the pig NCBI RefSeq mRNAs, and laid a foundation for blood-based high throughput transcriptomic assays in pigs and for advancing annotation of the pig genome.

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September 22, 2019

Isoform sequencing provides a more comprehensive view of the Panax ginseng transcriptome.

Korean ginseng (Panax ginseng C.A. Meyer) has been widely used for medicinal purposes and contains potent plant secondary metabolites, including ginsenosides. To obtain transcriptomic data that offers a more comprehensive view of functional genomics in P. ginseng, we generated genome-wide transcriptome data from four different P. ginseng tissues using PacBio isoform sequencing (Iso-Seq) technology. A total of 135,317 assembled transcripts were generated with an average length of 3.2 kb and high assembly completeness. Of those unigenes, 67.5% were predicted to be complete full-length (FL) open reading frames (ORFs) and exhibited a high gene annotation rate. Furthermore, we successfully identified unique full-length genes involved in triterpenoid saponin synthesis and plant hormonal signaling pathways, including auxin and cytokinin. Studies on the functional genomics of P. ginseng seedlings have confirmed the rapid upregulation of negative feed-back loops by auxin and cytokinin signaling cues. The conserved evolutionary mechanisms in the auxin and cytokinin canonical signaling pathways of P. ginseng are more complex than those in Arabidopsis thaliana. Our analysis also revealed a more detailed view of transcriptome-wide alternative isoforms for 88 genes. Finally, transposable elements (TEs) were also identified, suggesting transcriptional activity of TEs in P. ginseng. In conclusion, our results suggest that long-read, full-length or partial-unigene data with high-quality assemblies are invaluable resources as transcriptomic references in P. ginseng and can be used for comparative analyses in closely related medicinal plants.

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September 22, 2019

Genetic determinants of in vivo fitness and diet responsiveness in multiple human gut Bacteroides.

Libraries of tens of thousands of transposon mutants generated from each of four human gut Bacteroides strains, two representing the same species, were introduced simultaneously into gnotobiotic mice together with 11 other wild-type strains to generate a 15-member artificial human gut microbiota. Mice received one of two distinct diets monotonously, or both in different ordered sequences. Quantifying the abundance of mutants in different diet contexts allowed gene-level characterization of fitness determinants, niche, stability, and resilience and yielded a prebiotic (arabinoxylan) that allowed targeted manipulation of the community. The approach described is generalizable and should be useful for defining mechanisms critical for sustaining and/or approaches for deliberately reconfiguring the highly adaptive and durable relationship between the human gut microbiota and host in ways that promote wellness. Copyright © 2015, American Association for the Advancement of Science.

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