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September 22, 2019  |  

Advantages of genome sequencing by long-read sequencer using SMRT technology in medical area.

PacBio RS II is the first commercialized third-generation DNA sequencer able to sequence a single molecule DNA in real-time without amplification. PacBio RS II’s sequencing technology is novel and unique, enabling the direct observation of DNA synthesis by DNA polymerase. PacBio RS II confers four major advantages compared to other sequencing technologies: long read lengths, high consensus accuracy, a low degree of bias, and simultaneous capability of epigenetic characterization. These advantages surmount the obstacle of sequencing genomic regions such as high/low G+C, tandem repeat, and interspersed repeat regions. Moreover, PacBio RS II is ideal for whole genome sequencing, targeted sequencing, complex population analysis, RNA sequencing, and epigenetics characterization. With PacBio RS II, we have sequenced and analyzed the genomes of many species, from viruses to humans. Herein, we summarize and review some of our key genome sequencing projects, including full-length viral sequencing, complete bacterial genome and almost-complete plant genome assemblies, and long amplicon sequencing of a disease-associated gene region. We believe that PacBio RS II is not only an effective tool for use in the basic biological sciences but also in the medical/clinical setting.

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September 22, 2019  |  

CLK-dependent exon recognition and conjoined gene formation revealed with a novel small molecule inhibitor.

CDC-like kinase phosphorylation of serine/arginine-rich proteins is central to RNA splicing reactions. Yet, the genomic network of CDC-like kinase-dependent RNA processing events remains poorly defined. Here, we explore the connectivity of genomic CDC-like kinase splicing functions by applying graduated, short-exposure, pharmacological CDC-like kinase inhibition using a novel small molecule (T3) with very high potency, selectivity, and cell-based stability. Using RNA-Seq, we define CDC-like kinase-responsive alternative splicing events, the large majority of which monotonically increase or decrease with increasing CDC-like kinase inhibition. We show that distinct RNA-binding motifs are associated with T3 response in skipped exons. Unexpectedly, we observe dose-dependent conjoined gene transcription, which is associated with motif enrichment in the last and second exons of upstream and downstream partners, respectively. siRNA knockdown of CLK2-associated genes significantly increases conjoined gene formation. Collectively, our results reveal an unexpected role for CDC-like kinase in conjoined gene formation, via regulation of 3′-end processing and associated splicing factors.The phosphorylation of serine/arginine-rich proteins by CDC-like kinase is a central regulatory mechanism for RNA splicing reactions. Here, the authors synthesize a novel small molecule CLK inhibitor and map CLK-responsive alternative splicing events and discover an effect on conjoined gene transcription.

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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  |  

A workflow for studying specialized metabolism in nonmodel eukaryotic organisms

Eukaryotes contain a diverse tapestry of specialized metabolites, many of which are of significant pharmaceutical and industrial importance to humans. Nevertheless, exploration of specialized metabolic pathways underlying specific chemical traits in nonmodel eukaryotic organisms has been technically challenging and historically lagged behind that of the bacterial systems. Recent advances in genomics, metabolomics, phylogenomics, and synthetic biology now enable a new workflow for interrogating unknown specialized metabolic systems in nonmodel eukaryotic hosts with greater efficiency and mechanistic depth. This chapter delineates such workflow by providing a collection of state-of-the-art approaches and tools, ranging from multiomics-guided candidate gene identification to in vitro and in vivo functional and structural characterization of specialized metabolic enzymes. As already demonstrated by several recent studies, this new workflow opens up a gateway into the largely untapped world of natural product biochemistry in eukaryotes. © 2016 Elsevier Inc. All rights reserved.

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September 22, 2019  |  

Next-generation sequencing for pathogen detection and identification

Over the past decade, the field of genomics has seen such drastic improvements in sequencing chemistries that high-throughput sequencing, or next-generation sequencing (NGS), is being applied to generate data across many disciplines. NGS instruments are becoming less expensive, faster, and smaller, and therefore are being adopted in an increasing number of laboratories, including clinical laboratories. Thus far, clinical use of NGS has been mostly focused on the human genome, for purposes such as characterizing the molecular basis of cancer or for diagnosing and understanding the basis of rare genetic disorders. There are, however, an increasing number of examples whereby NGS is employed to discover novel pathogens, and these cases provide precedent for the use of NGS in microbial diagnostics. NGS has many advantages over traditional microbial diagnostic methods, such as unbiased rather than pathogen-specific protocols, ability to detect fastidious or non-culturable organisms, and ability to detect co-infections. One of the most impressive advantages of NGS is that it requires little or no prior knowledge of the pathogen, unlike many other diagnostic assays; therefore for pathogen discovery, NGS is very valuable. However, despite these advantages, there are challenges involved in implementing NGS for routine clinical microbiological diagnosis. We discuss these advantages and challenges in the context of recently described research studies.

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September 22, 2019  |  

The new world of isoform sequencing

Not too long ago, the life sciences community was still debating whether sequencers would ever overtake microarrays as the preferred means of measuring gene expression. Today, not only have sequencers become the standard workhorse for gene expression studies, but newer sequencing technology has delivered the ability to generate novel expression data even in the most well-characterized cells or organisms. Truly, it is a remarkable time for comprehensive studies of which genes are being transcribed, with the goal of providing functional insight into various biological processes. The key advantage sequencing holds over microarrays is its ability to deeply survey an entire transcriptome, while microarrays are limited to interrogating known genes using probes designed from a reference genome assembly. As next-generation sequencing became more affordable, scientists were eager to switch to this approach, which became known as RNA sequencing or simply RNA-seq. © Mary Ann Liebert, Inc.

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September 22, 2019  |  

Metataxonomic and metagenomic approaches vs. culture-based techniques for clinical pathology.

Diagnoses that are both timely and accurate are critically important for patients with life-threatening or drug resistant infections. Technological improvements in High-Throughput Sequencing (HTS) have led to its use in pathogen detection and its application in clinical diagnoses of infectious diseases. The present study compares two HTS methods, 16S rRNA marker gene sequencing (metataxonomics) and whole metagenomic shotgun sequencing (metagenomics), in their respective abilities to match the same diagnosis as traditional culture methods (culture inference) for patients with ventilator associated pneumonia (VAP). The metagenomic analysis was able to produce the same diagnosis as culture methods at the species-level for five of the six samples, while the metataxonomic analysis was only able to produce results with the same species-level identification as culture for two of the six samples. These results indicate that metagenomic analyses have the accuracy needed for a clinical diagnostic tool, but full integration in diagnostic protocols is contingent on technological improvements to decrease turnaround time and lower costs.

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September 22, 2019  |  

Proteogenomic analysis reveals alternative splicing and translation as part of the abscisic acid response in Arabidopsis seedlings.

In eukaryotes, mechanisms such as alternative splicing (AS) and alternative translation initiation (ATI) contribute to organismal protein diversity. Specifically, splicing factors play crucial roles in responses to environment and development cues; however, the underlying mechanisms are not well investigated in plants. Here, we report the parallel employment of short-read RNA sequencing, single molecule long-read sequencing and proteomic identification to unravel AS isoforms and previously unannotated proteins in response to abscisic acid (ABA) treatment. Combining the data from the two sequencing methods, approximately 83.4% of intron-containing genes were alternatively spliced. Two AS types, which are referred to as alternative first exon (AFE) and alternative last exon (ALE), were more abundant than intron retention (IR); however, by contrast to AS events detected under normal conditions, differentially expressed AS isoforms were more likely to be translated. ABA extensively affects the AS pattern, indicated by the increasing number of non-conventional splicing sites. This work also identified thousands of unannotated peptides and proteins by ATI based on mass spectrometry and a virtual peptide library deduced from both strands of coding regions within the Arabidopsis genome. The results enhance our understanding of AS and alternative translation mechanisms under normal conditions, and in response to ABA treatment.© 2017 The Authors The Plant Journal © 2017 John Wiley & Sons Ltd.

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September 22, 2019  |  

How far can mitochondrial DNA drive the disease?

Mitochondria are one of the dominant drivers for producing cellular energy to meet a large number of biological functions, of which the mitochondrial DNA (mtDNA) is the control center of energetic driving force and the dominant driver of mitochondrial molecular diversification. mtDNA transcription generates the necessary RNAs to regulate the extent and nature of mtRNA post-transcriptional modifications and the activity of nucleus-encoded enzymes. With a special focus on mtDNA, the current volume aims to overview the biology and structures of mtDNA, regulatory roles of mtDNA in lung diseases, or involvement of mtDNA in metabolism. We explore the significance of mtDNA sequencing, methylation, stability, and mutation in the pathogenesis of the diseases. Molecular mechanisms by which mtDNA contribute to the regulation of mitochondrial homeostasis and drug resistance are also discussed. We also point out the importance of mitochondrial ribosome, single cell biology, and gene editing in the understanding of the development of mitochondrial dysfunction in lung disease.

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September 22, 2019  |  

Interpreting microbial biosynthesis in the genomic age: Biological and practical considerations.

Genome mining has become an increasingly powerful, scalable, and economically accessible tool for the study of natural product biosynthesis and drug discovery. However, there remain important biological and practical problems that can complicate or obscure biosynthetic analysis in genomic and metagenomic sequencing projects. Here, we focus on limitations of available technology as well as computational and experimental strategies to overcome them. We review the unique challenges and approaches in the study of symbiotic and uncultured systems, as well as those associated with biosynthetic gene cluster (BGC) assembly and product prediction. Finally, to explore sequencing parameters that affect the recovery and contiguity of large and repetitive BGCs assembled de novo, we simulate Illumina and PacBio sequencing of the Salinispora tropica genome focusing on assembly of the salinilactam (slm) BGC.

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September 22, 2019  |  

LSCplus: a fast solution for improving long read accuracy by short read alignment.

The single molecule, real time (SMRT) sequencing technology of Pacific Biosciences enables the acquisition of transcripts from end to end due to its ability to produce extraordinarily long reads (>10 kb). This new method of transcriptome sequencing has been applied to several projects on humans and model organisms. However, the raw data from SMRT sequencing are of relatively low quality, with a random error rate of approximately 15 %, for which error correction using next-generation sequencing (NGS) short reads is typically necessary. Few tools have been designed that apply a hybrid sequencing approach that combines NGS and SMRT data, and the most popular existing tool for error correction, LSC, has computing resource requirements that are too intensive for most laboratory and research groups. These shortcomings severely limit the application of SMRT long reads for transcriptome analysis.Here, we report an improved tool (LSCplus) for error correction with the LSC program as a reference. LSCplus overcomes the disadvantage of LSC’s time consumption and improves quality. Only 1/3-1/4 of the time and 1/20-1/25 of the error correction time is required using LSCplus compared with that required for using LSC.LSCplus is freely available at http://www.herbbol.org:8001/lscplus/ . Sample calculations are provided illustrating the precision and efficiency of this method regarding error correction and isoform detection.

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September 22, 2019  |  

Mode of action and heterologous expression of the natural product antibiotic vancoresmycin.

Antibiotics that interfere with the bacterial cytoplasmic membrane have long-term potential for the treatment of infectious diseases as this mode of action is anticipated to result in low resistance frequency. Vancoresmycin is an understudied natural product antibiotic consisting of a terminal tetramic acid moiety fused to a linear, highly oxygenated, stereochemically complex polyketide chain. Vancoresmycin shows minimum inhibitory concentrations (MICs) from 0.125 to 2 µg/mL against a range of clinically relevant, antibiotic-resistant Gram-positive bacteria. Through a comprehensive mode-of-action study, utilizing Bacillus subtilis reporter strains, DiSC3(5) depolarization assays, and fluorescence microscopy, we have shown that vancoresmycin selectively targets the cytoplasmic membrane of Gram-positive bacteria via a non-pore-forming, concentration-dependent depolarization mechanism. Whole genome sequencing of the producing strain allowed identification of the 141 kbp gene cluster encoding for vancoresmycin biosynthesis and a preliminary model for its biosynthesis. The size and complex structure of vancoresmycin could confound attempts to generate synthetic analogues. To overcome this problem and facilitate future studies, we identified, cloned, and expressed the 141 kbp biosynthetic gene cluster in Streptomyces coelicolor M1152. Elucidation of the mode-of-action of vancoresmycin, together with the heterologous expression system, will greatly facilitate further studies of this and related molecules.

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September 22, 2019  |  

Vertebrate genome evolution in the light of fish cytogenomics and rDNAomics.

To understand the cytogenomic evolution of vertebrates, we must first unravel the complex genomes of fishes, which were the first vertebrates to evolve and were ancestors to all other vertebrates. We must not forget the immense time span during which the fish genomes had to evolve. Fish cytogenomics is endowed with unique features which offer irreplaceable insights into the evolution of the vertebrate genome. Due to the general DNA base compositional homogeneity of fish genomes, fish cytogenomics is largely based on mapping DNA repeats that still represent serious obstacles in genome sequencing and assembling, even in model species. Localization of repeats on chromosomes of hundreds of fish species and populations originating from diversified environments have revealed the biological importance of this genomic fraction. Ribosomal genes (rDNA) belong to the most informative repeats and in fish, they are subject to a more relaxed regulation than in higher vertebrates. This can result in formation of a literal ‘rDNAome’ consisting of more than 20,000 copies with their high proportion employed in extra-coding functions. Because rDNA has high rates of transcription and recombination, it contributes to genome diversification and can form reproductive barrier. Our overall knowledge of fish cytogenomics grows rapidly by a continuously increasing number of fish genomes sequenced and by use of novel sequencing methods improving genome assembly. The recently revealed exceptional compositional heterogeneity in an ancient fish lineage (gars) sheds new light on the compositional genome evolution in vertebrates generally. We highlight the power of synergy of cytogenetics and genomics in fish cytogenomics, its potential to understand the complexity of genome evolution in vertebrates, which is also linked to clinical applications and the chromosomal backgrounds of speciation. We also summarize the current knowledge on fish cytogenomics and outline its main future avenues.

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September 22, 2019  |  

Sooty mangabey genome sequence provides insight into AIDS resistance in a natural SIV host.

In contrast to infections with human immunodeficiency virus (HIV) in humans and simian immunodeficiency virus (SIV) in macaques, SIV infection of a natural host, sooty mangabeys (Cercocebus atys), is non-pathogenic despite high viraemia. Here we sequenced and assembled the genome of a captive sooty mangabey. We conducted genome-wide comparative analyses of transcript assemblies from C. atys and AIDS-susceptible species, such as humans and macaques, to identify candidates for host genetic factors that influence susceptibility. We identified several immune-related genes in the genome of C. atys that show substantial sequence divergence from macaques or humans. One of these sequence divergences, a C-terminal frameshift in the toll-like receptor-4 (TLR4) gene of C. atys, is associated with a blunted in vitro response to TLR-4 ligands. In addition, we found a major structural change in exons 3-4 of the immune-regulatory protein intercellular adhesion molecule 2 (ICAM-2); expression of this variant leads to reduced cell surface expression of ICAM-2. These data provide a resource for comparative genomic studies of HIV and/or SIV pathogenesis and may help to elucidate the mechanisms by which SIV-infected sooty mangabeys avoid AIDS.

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