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September 22, 2019

Combination of novel and public RNA-seq datasets to generate an mRNA expression atlas for the domestic chicken.

The domestic chicken (Gallus gallus) is widely used as a model in developmental biology and is also an important livestock species. We describe a novel approach to data integration to generate an mRNA expression atlas for the chicken spanning major tissue types and developmental stages, using a diverse range of publicly-archived RNA-seq datasets and new data derived from immune cells and tissues.Randomly down-sampling RNA-seq datasets to a common depth and quantifying expression against a reference transcriptome using the mRNA quantitation tool Kallisto ensured that disparate datasets explored comparable transcriptomic space. The network analysis tool Graphia was used to extract clusters of co-expressed genes from the resulting expression atlas, many of which were tissue or cell-type restricted, contained transcription factors that have previously been implicated in their regulation, or were otherwise associated with biological processes, such as the cell cycle. The atlas provides a resource for the functional annotation of genes that currently have only a locus ID. We cross-referenced the RNA-seq atlas to a publicly available embryonic Cap Analysis of Gene Expression (CAGE) dataset to infer the developmental time course of organ systems, and to identify a signature of the expansion of tissue macrophage populations during development.Expression profiles obtained from public RNA-seq datasets – despite being generated by different laboratories using different methodologies – can be made comparable to each other. This meta-analytic approach to RNA-seq can be extended with new datasets from novel tissues, and is applicable to any species.


September 22, 2019

Metagenomic binning of a marine sponge microbiome reveals unity in defense but metabolic specialization.

Marine sponges are ancient metazoans that are populated by distinct and highly diverse microbial communities. In order to obtain deeper insights into the functional gene repertoire of the Mediterranean sponge Aplysina aerophoba, we combined Illumina short-read and PacBio long-read sequencing followed by un-targeted metagenomic binning. We identified a total of 37 high-quality bins representing 11 bacterial phyla and two candidate phyla. Statistical comparison of symbiont genomes with selected reference genomes revealed a significant enrichment of genes related to bacterial defense (restriction-modification systems, toxin-antitoxin systems) as well as genes involved in host colonization and extracellular matrix utilization in sponge symbionts. A within-symbionts genome comparison revealed a nutritional specialization of at least two symbiont guilds, where one appears to metabolize carnitine and the other sulfated polysaccharides, both of which are abundant molecules in the sponge extracellular matrix. A third guild of symbionts may be viewed as nutritional generalists that perform largely the same metabolic pathways but lack such extraordinary numbers of the relevant genes. This study characterizes the genomic repertoire of sponge symbionts at an unprecedented resolution and it provides greater insights into the molecular mechanisms underlying microbial-sponge symbiosis.


September 22, 2019

Genomic microdiversity of Bifidobacterium pseudocatenulatum underlying differential strain-level responses to dietary carbohydrate intervention.

The genomic basis of the response to dietary intervention of human gut beneficial bacteria remains elusive, which hinders precise manipulation of the microbiota for human health. After receiving a dietary intervention enriched with nondigestible carbohydrates for 105 days, a genetically obese child with Prader-Willi syndrome lost 18.4% of his body weight and showed significant improvement in his bioclinical parameters. We obtained five isolates (C1, C15, C55, C62, and C95) of one of the most abundantly promoted beneficial species, Bifidobacterium pseudocatenulatum, from a postintervention fecal sample. Intriguingly, these five B. pseudocatenulatum strains showed differential responses during the dietary intervention. Two strains were largely unaffected, while the other three were promoted to different extents by the changes in dietary carbohydrate resources. The differential responses of these strains were consistent with their functional clustering based on the COGs (Clusters of Orthologous Groups), including those involved with the ABC-type sugar transport systems, suggesting that the strain-specific genomic variations may have contributed to the niche adaption. Particularly, B. pseudocatenulatum C15, which had the most diverse types and highest gene copy numbers of carbohydrate-active enzymes targeting plant polysaccharides, had the highest abundance after the dietary intervention. These studies show the importance of understanding genomic diversity of specific members of the gut microbiota if precise nutrition approaches are to be realized.IMPORTANCE The manipulation of the gut microbiota via dietary approaches is a promising option for improving human health. Our findings showed differential responses of multiple B. pseudocatenulatum strains isolated from the same habitat to the dietary intervention, as well as strain-specific correlations with bioclinical parameters of the host. The comparative genomics revealed a genome-level microdiversity of related functional genes, which may have contributed to these differences. These results highlight the necessity of understanding strain-level differences if precise manipulation of gut microbiota through dietary approaches is to be realized. Copyright © 2017 Wu et al.


September 22, 2019

Novel molecules lncRNAs, tRFs and circRNAs deciphered from next-generation sequencing/RNA sequencing: computational databases and tools.

Powerful next-generation sequencing (NGS) technologies, more specifically RNA sequencing (RNA-seq), have been pivotal toward the detection and analysis and hypotheses generation of novel biomolecules, long noncoding RNAs (lncRNAs), tRNA-derived fragments (tRFs) and circular RNAs (circRNAs). Experimental validation of the occurrence of these biomolecules inside the cell has been reported. Their differential expression and functionally important role in several cancers types as well as other diseases such as Alzheimer’s and cardiovascular diseases have garnered interest toward further studies in this research arena. In this review, starting from a brief relevant introduction to NGS and RNA-seq and the expression and role of lncRNAs, tRFs and circRNAs in cancer, we have comprehensively analyzed the current landscape of databases developed and computational software used for analyses and visualization for this emerging and highly interesting field of these novel biomolecules. Our review will help the end users and research investigators gain information on the existing databases and tools as well as an understanding of the specific features which these offer. This will be useful for the researchers in their proper usage thereby guiding them toward novel hypotheses generation and saving time and costs involved in extensive experimental processes in these three different novel functional RNAs.© The Author 2017. Published by Oxford University Press. All rights reserved. For permissions, please email: journals.permissions@oup.com.


September 22, 2019

Single-molecule long-read sequencing facilitates shrimp transcriptome research.

Although shrimp are of great economic importance, few full-length shrimp transcriptomes are available. Here, we used Pacific Biosciences single-molecule real-time (SMRT) long-read sequencing technology to generate transcripts from the Pacific white shrimp (Litopenaeus vannamei). We obtained 322,600 full-length non-chimeric reads, from which we generated 51,367 high-quality unique full-length transcripts. We corrected errors in the SMRT sequences by comparison with Illumina-produced short reads. We successfully annotated 81.72% of all unique SMRT transcripts against the NCBI non-redundant database, 58.63% against Swiss-Prot, 45.38% against Gene Ontology, 32.57% against Clusters of Orthologous Groups of proteins (COG), and 47.83% against Kyoto Encyclopedia of Genes and Genomes (KEGG) databases. Across all transcripts, we identified 3,958 long non-coding RNAs (lncRNAs) and 80,650 simple sequence repeats (SSRs). Our study provides a rich set of full-length cDNA sequences for L. vannamei, which will greatly facilitate shrimp transcriptome research.


September 22, 2019

A survey of the sorghum transcriptome using single-molecule long reads.

Alternative splicing and alternative polyadenylation (APA) of pre-mRNAs greatly contribute to transcriptome diversity, coding capacity of a genome and gene regulatory mechanisms in eukaryotes. Second-generation sequencing technologies have been extensively used to analyse transcriptomes. However, a major limitation of short-read data is that it is difficult to accurately predict full-length splice isoforms. Here we sequenced the sorghum transcriptome using Pacific Biosciences single-molecule real-time long-read isoform sequencing and developed a pipeline called TAPIS (Transcriptome Analysis Pipeline for Isoform Sequencing) to identify full-length splice isoforms and APA sites. Our analysis reveals transcriptome-wide full-length isoforms at an unprecedented scale with over 11,000 novel splice isoforms. Additionally, we uncover APA of ~11,000 expressed genes and more than 2,100 novel genes. These results greatly enhance sorghum gene annotations and aid in studying gene regulation in this important bioenergy crop. The TAPIS pipeline will serve as a useful tool to analyse Iso-Seq data from any organism.


September 22, 2019

Functional mitochondria in health and disease.

The ability to rapidly adapt cellular bioenergetic capabilities to meet rapidly changing environmental conditions is mandatory for normal cellular function and for cancer progression. Any loss of this adaptive response has the potential to compromise cellular function and render the cell more susceptible to external stressors such as oxidative stress, radiation, chemotherapeutic drugs, and hypoxia. Mitochondria play a vital role in bioenergetic and biosynthetic pathways and can rapidly adjust to meet the metabolic needs of the cell. Increased demand is met by mitochondrial biogenesis and fusion of individual mitochondria into dynamic networks, whereas a decrease in demand results in the removal of superfluous mitochondria through fission and mitophagy. Effective communication between nucleus and mitochondria (mito-nuclear cross talk), involving the generation of different mitochondrial stress signals as well as the nuclear stress response pathways to deal with these stressors, maintains bioenergetic homeostasis under most conditions. However, when mitochondrial DNA (mtDNA) mutations accumulate and mito-nuclear cross talk falters, mitochondria fail to deliver critical functional outputs. Mutations in mtDNA have been implicated in neuromuscular and neurodegenerative mitochondriopathies and complex diseases such as diabetes, cardiovascular diseases, gastrointestinal disorders, skin disorders, aging, and cancer. In some cases, drastic measures such as acquisition of new mitochondria from donor cells occurs to ensure cell survival. This review starts with a brief discussion of the evolutionary origin of mitochondria and summarizes how mutations in mtDNA lead to mitochondriopathies and other degenerative diseases. Mito-nuclear cross talk, including various stress signals generated by mitochondria and corresponding stress response pathways activated by the nucleus are summarized. We also introduce and discuss a small family of recently discovered hormone-like mitopeptides that modulate body metabolism. Under conditions of severe mitochondrial stress, mitochondria have been shown to traffic between cells, replacing mitochondria in cells with damaged and malfunctional mtDNA. Understanding the processes involved in cellular bioenergetics and metabolic adaptation has the potential to generate new knowledge that will lead to improved treatment of many of the metabolic, degenerative, and age-related inflammatory diseases that characterize modern societies.


September 22, 2019

Isoform evolution in primates through independent combination of alternative RNA processing events.

Recent RNA-seq technology revealed thousands of splicing events that are under rapid evolution in primates, whereas the reliability of these events, as well as their combination on the isoform level, have not been adequately addressed due to its limited sequencing length. Here, we performed comparative transcriptome analyses in human and rhesus macaque cerebellum using single molecule long-read sequencing (Iso-seq) and matched RNA-seq. Besides 359 million RNA-seq reads, 4,165,527 Iso-seq reads were generated with a mean length of 14,875?bp, covering 11,466 human genes, and 10,159 macaque genes. With Iso-seq data, we substantially expanded the repertoire of alternative RNA processing events in primates, and found that intron retention and alternative polyadenylation are surprisingly more prevalent in primates than previously estimated. We then investigated the combinatorial mode of these alternative events at the whole-transcript level, and found that the combination of these events is largely independent along the transcript, leading to thousands of novel isoforms missed by current annotations. Notably, these novel isoforms are selectively constrained in general, and 1,119 isoforms have even higher expression than the previously annotated major isoforms in human, indicating that the complexity of the human transcriptome is still significantly underestimated. Comparative transcriptome analysis further revealed 502 genes encoding selectively constrained, lineage-specific isoforms in human but not in rhesus macaque, linking them to some lineage-specific functions. Overall, we propose that the independent combination of alternative RNA processing events has contributed to complex isoform evolution in primates, which provides a new foundation for the study of phenotypic difference among primates.© The Author 2017. Published by Oxford University Press on behalf of the Society for Molecular Biology and Evolution.


September 22, 2019

Plasmodium knowlesi: a superb in vivo nonhuman primate model of antigenic variation in malaria.

Antigenic variation in malaria was discovered in Plasmodium knowlesi studies involving longitudinal infections of rhesus macaques (M. mulatta). The variant proteins, known as the P. knowlesi Schizont Infected Cell Agglutination (SICA) antigens and the P. falciparum Erythrocyte Membrane Protein 1 (PfEMP1) antigens, expressed by the SICAvar and var multigene families, respectively, have been studied for over 30 years. Expression of the SICA antigens in P. knowlesi requires a splenic component, and specific antibodies are necessary for variant antigen switch events in vivo. Outstanding questions revolve around the role of the spleen and the mechanisms by which the expression of these variant antigen families are regulated. Importantly, the longitudinal dynamics and molecular mechanisms that govern variant antigen expression can be studied with P. knowlesi infection of its mammalian and vector hosts. Synchronous infections can be initiated with established clones and studied at multi-omic levels, with the benefit of computational tools from systems biology that permit the integration of datasets and the design of explanatory, predictive mathematical models. Here we provide an historical account of this topic, while highlighting the potential for maximizing the use of P. knowlesi – macaque model systems and summarizing exciting new progress in this area of research.


September 22, 2019

Complete genome sequence of Sphingobium baderi DE-13, an alkyl-substituted aniline-mineralizing bacterium.

Alkyl-substituted aniline is an important aniline derivative that may be associated with serious environmental risks. Previously, Sphingobium baderi DE-13, a bacterium that can mineralize alkyl substituted anilines such as 2,6-dimethylaniline, 2,6-diethylaniline, 2-methyl-6-ethylaniline, 2-methylaniline, and 2-ethylaniline, was isolated from active sludge. Here, we report the complete genome sequence of strain DE-13. It contains one circular chromosome and eight circular plasmids with total 4,583,422 bp and GC content of 62.41%. The reported and predicted genes involved in the catabolism of alkyl-substituted anilines are indicated. This study will provide insights into the bacterial catabolism of alkyl substituted anilines.


September 22, 2019

Genome characterization of oleaginous Aspergillus oryzae BCC7051: A potential fungal-based platform for lipid production.

The selected robust fungus, Aspergillus oryzae strain BCC7051 is of interest for biotechnological production of lipid-derived products due to its capability to accumulate high amount of intracellular lipids using various sugars and agro-industrial substrates. Here, we report the genome sequence of the oleaginous A. oryzae BCC7051. The obtained reads were de novo assembled into 25 scaffolds spanning of 38,550,958 bps with predicted 11,456 protein-coding genes. By synteny mapping, a large rearrangement was found in two scaffolds of A. oryzae BCC7051 as compared to the reference RIB40 strain. The genetic relationship between BCC7051 and other strains of A. oryzae in terms of aflatoxin production was investigated, indicating that the A. oryzae BCC7051 was categorized into group 2 nonaflatoxin-producing strain. Moreover, a comparative analysis of the structural genes focusing on the involvement in lipid metabolism among oleaginous yeast and fungi revealed the presence of multiple isoforms of metabolic enzymes responsible for fatty acid synthesis in BCC7051. The alternative routes of acetyl-CoA generation as oleaginous features and malate/citrate/pyruvate shuttle were also identified in this A. oryzae strain. The genome sequence generated in this work is a dedicated resource for expanding genome-wide study of microbial lipids at systems level, and developing the fungal-based platform for production of diversified lipids with commercial relevance.


September 22, 2019

Extensively drug-resistant Escherichia coli sequence type 1642 carrying an IncX3 plasmid containing the blaKPC-2 gene associated with transposon Tn4401a.

Extensively drug-resistant (XDR) Enterobacteriaceae carrying the bla(KPC) gene have emerged as a major global therapeutic concern. The purpose of this study was to analyze the complete sequences of plasmids from KPC-2 carbapenemase-producing XDR Escherichia coli sequence type (ST) 1642 isolates.We performed antimicrobial susceptibility testing, PCR, multilocus sequence typing (MLST), and whole-genome sequencing to characterize the plasmid-mediated KPC-2-producing E. coli clinical isolates.The isolates were resistant to most available antibiotics, including meropenem, ampicillin, ceftriaxone, gentamicin, and ciprofloxacin, but susceptible to tigecycline and colistin. The isolates were identified as the rare ST1642 by MLST. The isolates carried four plasmids: the first 69-kb conjugative IncX3 plasmid harbors bla(KPC-2) within a truncated Tn4401a transposon and bla(SHV-11) with duplicated conjugative elements. The second 142-kb plasmid with a multireplicon consisting of IncQ, IncFIA, and IncIB carries bla(TEM-1b) and two class 1 integrons. This plasmid also harbors a wide variety of additional antimicrobial resistance genes including aadA5, dfrA17, mph(A), sul1, tet(B), aac(3′)-IId, strA, strB, and sul2.The complete sequence analysis of plasmids from an XDR E. coli strain related to persistent infection showed the coexistence of a bla(KPC-2)-carrying IncX3-type plasmid and a class 1 integron-harboring multireplicon, suggesting its potential to cause outbreaks. Of additional clinical significance, the rare ST1642, identified in a cat, could constitute the source of human infection.


September 22, 2019

Fluorescently-tagged human eIF3 for single-molecule spectroscopy.

Human translation initiation relies on the combined activities of numerous ribosome-associated eukaryotic initiation factors (eIFs). The largest factor, eIF3, is an ~800 kDa multiprotein complex that orchestrates a network of interactions with the small 40S ribosomal subunit, other eIFs, and mRNA, while participating in nearly every step of initiation. How these interactions take place during the time course of translation initiation remains unclear. Here, we describe a method for the expression and affinity purification of a fluorescently-tagged eIF3 from human cells. The tagged eIF3 dodecamer is structurally intact, functions in cell-based assays, and interacts with the HCV IRES mRNA and the 40S-IRES complex in vitro. By tracking the binding of single eIF3 molecules to the HCV IRES RNA with a zero-mode waveguides-based instrument, we show that eIF3 samples both wild-type IRES and an IRES that lacks the eIF3-binding region, and that the high-affinity eIF3-IRES interaction is largely determined by slow dissociation kinetics. The application of single-molecule methods to more complex systems involving eIF3 may unveil dynamics underlying mRNA selection and ribosome loading during human translation initiation.© The Author(s) 2017. Published by Oxford University Press on behalf of Nucleic Acids Research.


September 22, 2019

Egg case silk gene sequences from Argiope spiders: Evidence for multiple loci and a loss of function between paralogs.

Spiders swath their eggs with silk to protect developing embryos and hatchlings. Egg case silks, like other fibrous spider silks, are primarily composed of proteins called spidroins (spidroin = spider-fibroin). Silks, and thus spidroins, are important throughout the lives of spiders, yet the evolution of spidroin genes has been relatively understudied. Spidroin genes are notoriously difficult to sequence because they are typically very long (= 10 kb of coding sequence) and highly repetitive. Here, we investigate the evolution of spider silk genes through long-read sequencing of Bacterial Artificial Chromosome (BAC) clones. We demonstrate that the silver garden spiderArgiope argentatahas multiple egg case spidroin loci with a loss of function at one locus. We also use degenerate PCR primers to search the genomic DNA of congeneric species and find evidence for multiple egg case spidroin loci in otherArgiopespiders. Comparative analyses show that these multiple loci are more similar at the nucleotide level within a species than between species. This pattern is consistent with concerted evolution homogenizing gene copies within a genome. More complicated explanations include convergent evolution or recent independent gene duplications within each species. Copyright © 2018 Chaw et al.


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