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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

SparseIso: a novel Bayesian approach to identify alternatively spliced isoforms from RNA-seq data.

Recent advances in high-throughput RNA sequencing (RNA-seq) technologies have made it possible to reconstruct the full transcriptome of various types of cells. It is important to accurately assemble transcripts or identify isoforms for an improved understanding of molecular mechanisms in biological systems.We have developed a novel Bayesian method, SparseIso, to reliably identify spliced isoforms from RNA-seq data. A spike-and-slab prior is incorporated into the Bayesian model to enforce the sparsity for isoform identification, effectively alleviating the problem of overfitting. A Gibbs sampling procedure is further developed to simultaneously identify and quantify transcripts from RNA-seq data. With the sampling approach, SparseIso estimates the joint distribution of all candidate transcripts, resulting in a significantly improved performance in detecting lowly expressed transcripts and multiple expressed isoforms of genes. Both simulation study and real data analysis have demonstrated that the proposed SparseIso method significantly outperforms existing methods for improved transcript assembly and isoform identification.The SparseIso package is available at http://github.com/henryxushi/SparseIso.xuan@vt.edu.Supplementary data are available at Bioinformatics online.© The Author (2017). Published by Oxford University Press. All rights reserved. For Permissions, please email: journals.permissions@oup.com

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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

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

Analysis of RNA base modification and structural rearrangement by single-molecule real-time detection of reverse transcription.

Zero-mode waveguides (ZMWs) are photonic nanostructures that create highly confined optical observation volumes, thereby allowing single-molecule-resolved biophysical studies at relatively high concentrations of fluorescent molecules. This principle has been successfully applied in single-molecule, real-time (SMRT®) DNA sequencing for the detection of DNA sequences and DNA base modifications. In contrast, RNA sequencing methods cannot provide sequence and RNA base modifications concurrently as they rely on complementary DNA (cDNA) synthesis by reverse transcription followed by sequencing of cDNA. Thus, information on RNA modifications is lost during the process of cDNA synthesis.Here we describe an application of SMRT technology to follow the activity of reverse transcriptase enzymes synthesizing cDNA on thousands of single RNA templates simultaneously in real time with single nucleotide turnover resolution using arrays of ZMWs. This method thereby obtains information from the RNA template directly. The analysis of the kinetics of the reverse transcriptase can be used to identify RNA base modifications, shown by example for N6-methyladenine (m6A) in oligonucleotides and in a specific mRNA extracted from total cellular mRNA. Furthermore, the real-time reverse transcriptase dynamics informs about RNA secondary structure and its rearrangements, as demonstrated on a ribosomal RNA and an mRNA template.Our results highlight the feasibility of studying RNA modifications and RNA structural rearrangements in ZMWs in real time. In addition, they suggest that technology can be developed for direct RNA sequencing provided that the reverse transcriptase is optimized to resolve homonucleotide stretches in RNA.

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September 22, 2019

cDNA library enrichment of full length transcripts for SMRT long read sequencing.

The utility of genome assemblies does not only rely on the quality of the assembled genome sequence, but also on the quality of the gene annotations. The Pacific Biosciences Iso-Seq technology is a powerful support for accurate eukaryotic gene model annotation as it allows for direct readout of full-length cDNA sequences without the need for noisy short read-based transcript assembly. We propose the implementation of the TeloPrime Full Length cDNA Amplification kit to the Pacific Biosciences Iso-Seq technology in order to enrich for genuine full-length transcripts in the cDNA libraries. We provide evidence that TeloPrime outperforms the commonly used SMARTer PCR cDNA Synthesis Kit in identifying transcription start and end sites in Arabidopsis thaliana. Furthermore, we show that TeloPrime-based Pacific Biosciences Iso-Seq can be successfully applied to the polyploid genome of bread wheat (Triticum aestivum) not only to efficiently annotate gene models, but also to identify novel transcription sites, gene homeologs, splicing isoforms and previously unidentified gene loci.

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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

Fine mapping and candidate gene identification of the genic male-sterile gene ms3 in cabbage 51S.

The ms3 gene responsible for a male-sterile phenotype in cabbage was mapped to a 187.4-kb genomic fragment. The gene BoTPD1, a homolog of Arabidopsis TPD1, was identified as a strong candidate gene. Cabbage 51S is a spontaneous male-sterile mutant. Phenotypic investigation revealed defects in anther cell differentiation, with failure to form the tapetum layer and complete abortion of microsporocytes before the tetrad stage. Genetic analysis indicated that this male sterility was controlled by a single recessive gene, ms3. Using an F2 population, we mapped ms3 to a 187.4-kb interval. BoTPD1 was identified as a candidate from this interval. Sequence analysis revealed an intronic 182-bp insertion in 51S that interrupted the conserved motif at the 5′ splicing site of the third intron, possibly resulting in a truncated transcript. Analyses of BoTPD1 homologous proteins revealed evolutionarily conserved roles in anther cell fate determination during reproductive development. RT-PCR showed that BoTPD1 was expressed in various tissues, excluding the root, and high expression levels were detected in anthers and buds. A BoTPD1-specific marker based on the 182-bp insertion cosegregated with male sterility and can be used for marker-assisted selection.

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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

Chromosome-level reference genome and alternative splicing atlas of moso bamboo (Phyllostachys edulis).

Bamboo is one of the most important nontimber forestry products worldwide. However, a chromosome-level reference genome is lacking, and an evolutionary view of alternative splicing (AS) in bamboo remains unclear despite emerging omics data and improved technologies.Here, we provide a chromosome-level de novo genome assembly of moso bamboo (Phyllostachys edulis) using additional abundance sequencing data and a Hi-C scaffolding strategy. The significantly improved genome is a scaffold N50 of 79.90 Mb, approximately 243 times longer than the previous version. A total of 51,074 high-quality protein-coding loci with intact structures were identified using single-molecule real-time sequencing and manual verification. Moreover, we provide a comprehensive AS profile based on the identification of 266,711 unique AS events in 25,225 AS genes by large-scale transcriptomic sequencing of 26 representative bamboo tissues using both the Illumina and Pacific Biosciences sequencing platforms. Through comparisons with orthologous genes in related plant species, we observed that the AS genes are concentrated among more conserved genes that tend to accumulate higher transcript levels and share less tissue specificity. Furthermore, gene family expansion, abundant AS, and positive selection were identified in crucial genes involved in the lignin biosynthetic pathway of moso bamboo.These fundamental studies provide useful information for future in-depth analyses of comparative genome and AS features. Additionally, our results highlight a global perspective of AS during evolution and diversification in bamboo.

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September 22, 2019

Gaining comprehensive biological insight into the transcriptome by performing a broad-spectrum RNA-seq analysis.

RNA-sequencing (RNA-seq) is an essential technique for transcriptome studies, hundreds of analysis tools have been developed since it was debuted. Although recent efforts have attempted to assess the latest available tools, they have not evaluated the analysis workflows comprehensively to unleash the power within RNA-seq. Here we conduct an extensive study analysing a broad spectrum of RNA-seq workflows. Surpassing the expression analysis scope, our work also includes assessment of RNA variant-calling, RNA editing and RNA fusion detection techniques. Specifically, we examine both short- and long-read RNA-seq technologies, 39 analysis tools resulting in ~120 combinations, and ~490 analyses involving 15 samples with a variety of germline, cancer and stem cell data sets. We report the performance and propose a comprehensive RNA-seq analysis protocol, named RNACocktail, along with a computational pipeline achieving high accuracy. Validation on different samples reveals that our proposed protocol could help researchers extract more biologically relevant predictions by broad analysis of the transcriptome.RNA-seq is widely used for transcriptome analysis. Here, the authors analyse a wide spectrum of RNA-seq workflows and present a comprehensive analysis protocol named RNACocktail as well as a computational pipeline leveraging the widely used tools for accurate RNA-seq analysis.

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September 22, 2019

Genome-wide identification and analysis of the ALTERNATIVE OXIDASE gene family in diploid and hexaploid wheat.

A comprehensive understanding of wheat responses to environmental stress will contribute to the long-term goal of feeding the planet. ALERNATIVE OXIDASE (AOX) genes encode proteins involved in a bypass of the electron transport chain and are also known to be involved in stress tolerance in multiple species. Here, we report the identification and characterization of the AOX gene family in diploid and hexaploid wheat. Four genes each were found in the diploid ancestors Triticum urartu, and Aegilops tauschii, and three in Aegilops speltoides. In hexaploid wheat (Triticum aestivum), 20 genes were identified, some with multiple splice variants, corresponding to a total of 24 proteins for those with observed transcription and translation. These proteins were classified as AOX1a, AOX1c, AOX1e or AOX1d via phylogenetic analysis. Proteins lacking most or all signature AOX motifs were assigned to putative regulatory roles. Analysis of protein-targeting sequences suggests mixed localization to the mitochondria and other organelles. In comparison to the most studied AOX from Trypanosoma brucei, there were amino acid substitutions at critical functional domains indicating possible role divergence in wheat or grasses in general. In hexaploid wheat, AOX genes were expressed at specific developmental stages as well as in response to both biotic and abiotic stresses such as fungal pathogens, heat and drought. These AOX expression patterns suggest a highly regulated and diverse transcription and expression system. The insights gained provide a framework for the continued and expanded study of AOX genes in wheat for stress tolerance through breeding new varieties, as well as resistance to AOX-targeted herbicides, all of which can ultimately be used synergistically to improve crop yield.

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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

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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