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September 22, 2019

The dynamic landscape of fission yeast meiosis alternative-splice isoforms.

Alternative splicing increases the diversity of transcriptomes and proteomes in metazoans. The extent to which alternative splicing is active and functional in unicellular organisms is less understood. Here, we exploit a single-molecule long-read sequencing technique and develop an open-source software program called SpliceHunter to characterize the transcriptome in the meiosis of fission yeast. We reveal 14,353 alternative splicing events in 17,669 novel isoforms at different stages of meiosis, including antisense and read-through transcripts. Intron retention is the major type of alternative splicing, followed by alternate “intron in exon.” Seven hundred seventy novel transcription units are detected; 53 of the predicted proteins show homology in other species and form theoretical stable structures. We report the complexity of alternative splicing along isoforms, including 683 intra-molecularly co-associated intron pairs. We compare the dynamics of novel isoforms based on the number of supporting full-length reads with those of annotated isoforms and explore the translational capacity and quality of novel isoforms. The evaluation of these factors indicates that the majority of novel isoforms are unlikely to be both condition-specific and translatable but consistent with the possibility of biologically functional novel isoforms. Moreover, the co-option of these unusual transcripts into newly born genes seems likely. Together, the results of this study highlight the diversity and dynamics at the isoform level in the sexual development of fission yeast. © 2017 Kuang et al.; Published by Cold Spring Harbor Laboratory Press.


September 22, 2019

De novo transcriptome assembly of the Chinese pearl barley, adlay, by full-length isoform and short-read RNA sequencing.

Adlay (Coix lacryma-jobi) is a tropical grass that has long been used in traditional Chinese medicine and is known for its nutritional benefits. Recent studies have shown that vitamin E compounds in adlay protect against chronic diseases such as cancer and heart disease. However, the molecular basis of adlay’s health benefits remains unknown. Here, we generated adlay gene sets by de novo transcriptome assembly using long-read isoform sequencing (Iso-Seq) and short-read RNA-Sequencing (RNA-Seq). The gene sets obtained from Iso-seq and RNA-seq contained 31,177 genes and 57,901 genes, respectively. We confirmed the validity of the assembled gene sets by experimentally analyzing the levels of prolamin and vitamin E biosynthesis-associated proteins in adlay plant tissues and seeds. We compared the screened adlay genes with known gene families from closely related plant species, such as rice, sorghum and maize. We also identified tissue-specific genes from the adlay leaf, root, and young and mature seed, and experimentally validated the differential expression of 12 randomly-selected genes. Our study of the adlay transcriptome will provide a valuable resource for genetic studies that can enhance adlay breeding programs in the future.


September 22, 2019

Transcriptome sequencing reveals thousands of novel long non-coding RNAs in B cell lymphoma.

Gene profiling of diffuse large B cell lymphoma (DLBCL) has revealed broad gene expression deregulation compared to normal B cells. While many studies have interrogated well known and annotated genes in DLBCL, none have yet performed a systematic analysis to uncover novel unannotated long non-coding RNAs (lncRNA) in DLBCL. In this study we sought to uncover these lncRNAs by examining RNA-seq data from primary DLBCL tumors and performed supporting analysis to identify potential role of these lncRNAs in DLBCL.We performed a systematic analysis of novel lncRNAs from the poly-adenylated transcriptome of 116 primary DLBCL samples. RNA-seq data were processed using de novo transcript assembly pipeline to discover novel lncRNAs in DLBCL. Systematic functional, mutational, cross-species, and co-expression analyses using numerous bioinformatics tools and statistical analysis were performed to characterize these novel lncRNAs.We identified 2,632 novel, multi-exonic lncRNAs expressed in more than one tumor, two-thirds of which are not expressed in normal B cells. Long read single molecule sequencing supports the splicing structure of many of these lncRNAs. More than one-third of novel lncRNAs are differentially expressed between the two major DLBCL subtypes, ABC and GCB. Novel lncRNAs are enriched at DLBCL super-enhancers, with a fraction of them conserved between human and dog lymphomas. We see transposable elements (TE) overlap in the exonic regions; particularly significant in the last exon of the novel lncRNAs suggest potential usage of cryptic TE polyadenylation signals. We identified highly co-expressed protein coding genes for at least 88 % of the novel lncRNAs. Functional enrichment analysis of co-expressed genes predicts a potential function for about half of novel lncRNAs. Finally, systematic structural analysis of candidate point mutations (SNVs) suggests that such mutations frequently stabilize lncRNA structures instead of destabilizing them.Discovery of these 2,632 novel lncRNAs in DLBCL significantly expands the lymphoma transcriptome and our analysis identifies potential roles of these lncRNAs in lymphomagenesis and/or tumor maintenance. For further studies, these novel lncRNAs also provide an abundant source of new targets for antisense oligonucleotide pharmacology, including shared targets between human and dog lymphomas.


September 22, 2019

Long read reference genome-free reconstruction of a full-length transcriptome from Astragalus membranaceus reveals transcript variants involved in bioactive compound biosynthesis.

Astragalus membranaceus, also known as Huangqi in China, is one of the most widely used medicinal herbs in Traditional Chinese Medicine. Traditional Chinese Medicine formulations from Astragalus membranaceus have been used to treat a wide range of illnesses, such as cardiovascular disease, type 2 diabetes, nephritis and cancers. Pharmacological studies have shown that immunomodulating, anti-hyperglycemic, anti-inflammatory, antioxidant and antiviral activities exist in the extract of Astragalus membranaceus. Therefore, characterising the biosynthesis of bioactive compounds in Astragalus membranaceus, such as Astragalosides, Calycosin and Calycosin-7-O-ß-d-glucoside, is of particular importance for further genetic studies of Astragalus membranaceus. In this study, we reconstructed the Astragalus membranaceus full-length transcriptomes from leaf and root tissues using PacBio Iso-Seq long reads. We identified 27 975 and 22 343 full-length unique transcript models in each tissue respectively. Compared with previous studies that used short read sequencing, our reconstructed transcripts are longer, and are more likely to be full-length and include numerous transcript variants. Moreover, we also re-characterised and identified potential transcript variants of genes involved in Astragalosides, Calycosin and Calycosin-7-O-ß-d-glucoside biosynthesis. In conclusion, our study provides a practical pipeline to characterise the full-length transcriptome for species without a reference genome and a useful genomic resource for exploring the biosynthesis of active compounds in Astragalus membranaceus.


September 22, 2019

Convergent evolution driven by rifampin exacerbates the global burden of drug-resistant Staphylococcus aureus.

Mutations in the beta-subunit of bacterial RNA polymerase (RpoB) cause resistance to rifampin (Rifr), a critical antibiotic for treatment of multidrug-resistantStaphylococcus aureus.In vitrostudies have shown that RpoB mutations confer decreased susceptibility to other antibiotics, but the clinical relevance is unknown. Here, by analyzing 7,099S. aureusgenomes, we demonstrate that the most prevalent RpoB mutations promote clinically relevant phenotypic plasticity resulting in the emergence of stableS. aureuslineages, associated with increased risk of therapeutic failure through generation of small-colony variants (SCVs) and coresistance to last-line antimicrobial agents. We found eight RpoB mutations that accounted for 93% (469/505) of the total number of Rifrmutations. The most frequently selected amino acid substitutions affecting residue 481 (H481N/Y) were associated with worldwide expansions of Rifrclones spanning decades. Recreating the H481N/Y mutations confirmed no impact onS. aureusgrowth, but the H481N mutation promoted the emergence of a subpopulation of stable RifrSCVs with reduced susceptibility to vancomycin and daptomycin. Recreating the other frequent RpoB mutations showed similar impacts on resistance to these last-line agents. We found that 86% of all Rifrisolates in our global sample carried the mutations promoting cross-resistance to vancomycin and 52% to both vancomycin and daptomycin. As four of the most frequent RpoB mutations confer only low-level Rifr, equal to or below some international breakpoints, we recommend decreasing these breakpoints and reconsidering the appropriate use of rifampin to reduce the fixation and spread of these clinically deleterious mutations. IMPORTANCE Increasing antibiotic resistance in the major human pathogenStaphylococcus aureusis threatening the ability to treat patients with these infections. Recent laboratory studies suggest that mutations in the gene commonly associated with rifampin resistance may also impact susceptibility to other last-line antibiotics inS. aureus; however, the overall frequency and clinical impact of these mutations are unknown. By mining a global collection of clinicalS. aureusgenomes and by mutagenesis experiments, this work reveals that common rifampin-inducedrpoBmutations promote phenotypic plasticity that has led to the global emergence of stable, multidrug-resistantS. aureuslineages that are associated with increased risk of therapeutic failure through coresistance to other last-line antimicrobials. We recommend decreasing susceptibility breakpoints for rifampin to allow phenotypic detection of criticalrpoBmutations conferring low resistance to rifampin and reconsidering the appropriate use of rifampin to reduce the fixation and spread of these deleterious mutations globally.


September 22, 2019

High genetic plasticity in multidrug-resistant sequence type 3-IncHI2 plasmids revealed by sequence comparison and phylogenetic analysis.

We report a novel fusion plasmid, pP2-3T, cointegrating sequence type 3 (ST3)-IncHI2 with an IncFII plasmid backbone mediating multidrug resistance (MDR) and virulence. Phylogenetic analysis and comparative genomics revealed that pP2-3T and other MDR ST3-IncHI2 plasmids clustered together, representing a unique IncHI2 lineage that exhibited high conservation in backbones of plasmids but possessed highly genetic plasticity in various regions by acquiring numerous antibiotic resistance genes and fusing with other plasmids. Surveillance studies should be performed to monitor multiresistance IncHI2 plasmids among Enterobacteriaceae. Copyright © 2018 American Society for Microbiology.


September 22, 2019

Complete genome sequence and genomic characterization of Lactobacillus acidophilus LA1 (11869BP).

Our body has natural defense systems to protect against potentially harmful microbes, including the physical and chemical barriers of the intestinal epithelium (Corfield et al., 2000). The physical barrier of the intestinal epithelium protects the host against pathogenic microbes (Anderson et al., 1993), and the intestinal mucosa coated with mucus excretes pathogens from the intestinal tract (Corfield et al., 2000).


September 22, 2019

CliqueSNV: Scalable reconstruction of intra-host viral populations from NGS reads

Highly mutable RNA viruses such as influenza A virus, human immunodeficiency virus and hepatitis C virus exist in infected hosts as highly heterogeneous populations of closely related genomic variants. The presence of low-frequency variants with few mutations with respect to major strains may result in an immune escape, emergence of drug resistance, and an increase of virulence and infectivity. Next-generation sequencing technologies permit detection of sample intra-host viral population at extremely great depth, thus providing an opportunity to access low-frequency variants. Long read lengths offered by single-molecule sequencing technologies allow all viral variants to be sequenced in a single pass. However, high sequencing error rates limit the ability to study heterogeneous viral populations composed of rare, closely related variants. In this article, we present CliqueSNV, a novel reference-based method for reconstruction of viral variants from NGS data. It efficiently constructs an allele graph based on linkage between single nucleotide variations and identifies true viral variants by merging cliques of that graph using combinatorial optimization techniques. The new method outperforms existing methods in both accuracy and running time on experimental and simulated NGS data for titrated levels of known viral variants. For PacBio reads, it accurately reconstructs variants with frequency as low as 0.1%. For Illumina reads, it fully reconstructs main variants. The open source implementation of CliqueSNV is freely available for download at https://github.com/vyacheslav-tsivina/CliqueSNV


September 22, 2019

Ginseng Genome Database: an open-access platform for genomics of Panax ginseng.

The ginseng (Panax ginseng C.A. Meyer) is a perennial herbaceous plant that has been used in traditional oriental medicine for thousands of years. Ginsenosides, which have significant pharmacological effects on human health, are the foremost bioactive constituents in this plant. Having realized the importance of this plant to humans, an integrated omics resource becomes indispensable to facilitate genomic research, molecular breeding and pharmacological study of this herb.The first draft genome sequences of P. ginseng cultivar “Chunpoong” were reported recently. Here, using the draft genome, transcriptome, and functional annotation datasets of P. ginseng, we have constructed the Ginseng Genome Database http://ginsengdb.snu.ac.kr /, the first open-access platform to provide comprehensive genomic resources of P. ginseng. The current version of this database provides the most up-to-date draft genome sequence (of approximately 3000 Mbp of scaffold sequences) along with the structural and functional annotations for 59,352 genes and digital expression of genes based on transcriptome data from different tissues, growth stages and treatments. In addition, tools for visualization and the genomic data from various analyses are provided. All data in the database were manually curated and integrated within a user-friendly query page.This database provides valuable resources for a range of research fields related to P. ginseng and other species belonging to the Apiales order as well as for plant research communities in general. Ginseng genome database can be accessed at http://ginsengdb.snu.ac.kr /.


September 22, 2019

The genome of the marine medaka Oryzias melastigma.

Marine medaka (Oryzias melastigma) is considered to be a useful fish model for marine and estuarine ecotoxicology studies and has good potential for field-based population genomics because of its geographical distribution in Asian estuarine and coastal areas. In this study, we present the first whole-genome draft of O. melastigma. The genome assembly consists of 8,602 scaffolds (N50 = 23.737 Mb) and a total genome length of 779.4 Mb. A total of 23,528 genes were predicted, and 12,670 gene families shared with three teleost species (Japanese medaka, mangrove killifish and zebrafish) were identified. Genome analyses revealed that the O. melastigma genome is highly heterozygous and contains a large number of repeat sequences. This assembly represents a useful genomic resource for fish scientists.© 2018 John Wiley & Sons Ltd.


September 22, 2019

De novo genome assembly of the red silk cotton tree (Bombax ceiba).

Bombax ceiba L. (the red silk cotton tree) is a large deciduous tree that is distributed in tropical and sub-tropical Asia as well as northern Australia. It has great economic and ecological importance, with several applications in industry and traditional medicine in many Asian countries. To facilitate further utilization of this plant resource, we present here the draft genome sequence for B. ceiba.We assembled a relatively intact genome of B. ceiba by using PacBio single-molecule sequencing and BioNano optical mapping technologies. The final draft genome is approximately 895 Mb long, with contig and scaffold N50 sizes of 1.0 Mb and 2.06 Mb, respectively.The high-quality draft genome assembly of B. ceiba will be a valuable resource enabling further genetic improvement and more effective use of this tree species.


September 22, 2019

In vitro DNA SCRaMbLE.

The power of synthetic biology has enabled the expression of heterologous pathways in cells, as well as genome-scale synthesis projects. The complexity of biological networks makes rational de novo design a grand challenge. Introducing features that confer genetic flexibility is a powerful strategy for downstream engineering. Here we develop an in vitro method of DNA library construction based on structural variation to accomplish this goal. The “in vitro SCRaMbLE system” uses Cre recombinase mixed in a test tube with purified DNA encoding multiple loxPsym sites. Using a ß-carotene pathway designed for expression in yeast as an example, we demonstrate top-down and bottom-up in vitro SCRaMbLE, enabling optimization of biosynthetic pathway flux via the rearrangement of relevant transcription units. We show that our system provides a straightforward way to correlate phenotype and genotype and is potentially amenable to biochemical optimization in ways that the in vivo system cannot achieve.


September 22, 2019

The genome of Artemisia annua provides insight into the evolution of Asteraceae family and artemisinin biosynthesis.

Artemisia annua, commonly known as sweet wormwood or Qinghao, is a shrub native to China and has long been used for medicinal purposes. A. annua is now cultivated globally as the only natural source of a potent anti-malarial compound, artemisinin. Here, we report a high-quality draft assembly of the 1.74-gigabase genome of A. annua, which is highly heterozygous, rich in repetitive sequences, and contains 63 226 protein-coding genes, one of the largest numbers among the sequenced plant species. We found that, as one of a few sequenced genomes in the Asteraceae, the A. annua genome contains a large number of genes specific to this large angiosperm clade. Notably, the expansion and functional diversification of genes encoding enzymes involved in terpene biosynthesis are consistent with the evolution of the artemisinin biosynthetic pathway. We further revealed by transcriptome profiling that A. annua has evolved the sophisticated transcriptional regulatory networks underlying artemisinin biosynthesis. Based on comprehensive genomic and transcriptomic analyses we generated transgenic A. annua lines producing high levels of artemisinin, which are now ready for large-scale production and thereby will help meet the challenge of increasing global demand of artemisinin. Copyright © 2018 The Author. Published by Elsevier Inc. All rights reserved.


September 22, 2019

Improved de novo genome assembly and analysis of the Chinese cucurbit Siraitia grosvenorii, also known as monk fruit or luo-han-guo.

Luo-han-guo (Siraitia grosvenorii), also called monk fruit, is a member of the Cucurbitaceae family. Monk fruit has become an important area for research because of the pharmacological and economic potential of its noncaloric, extremely sweet components (mogrosides). It is also commonly used in traditional Chinese medicine for the treatment of lung congestion, sore throat, and constipation. Recently, a single reference genome became available for monk fruit, assembled from 36.9x genome coverage reads via Illumina sequencing platforms. This genome assembly has a relatively short (34.2 kb) contig N50 length and lacks integrated annotations. These drawbacks make it difficult to use as a reference in assembling transcriptomes and discovering novel functional genes.Here, we offer a new high-quality draft of the S. grosvenorii genome assembled using 31 Gb (~73.8x) long single molecule real time sequencing reads and polished with ~50 Gb Illumina paired-end reads. The final genome assembly is approximately 469.5 Mb, with a contig N50 length of 432,384 bp, representing a 12.6-fold improvement. We further annotated 237.3 Mb of repetitive sequence and 30,565 consensus protein coding genes with combined evidence. Phylogenetic analysis showed that S. grosvenorii diverged from members of the Cucurbitaceae family approximately 40.9 million years ago. With comprehensive transcriptomic analysis and differential expression testing, we identified 4,606 up-regulated genes in the early fruit compared to the leaf, a number of which were linked to metabolic pathways regulating fruit development and ripening.The availability of this new monk fruit genome assembly, as well as the annotations, will facilitate the discovery of new functional genes and the genetic improvement of monk fruit.


September 22, 2019

N6-methyladenine DNA modification in the human genome.

DNA N6-methyladenine (6mA) modification is the most prevalent DNA modification in prokaryotes, but whether it exists in human cells and whether it plays a role in human diseases remain enigmatic. Here, we showed that 6mA is extensively present in the human genome, and we cataloged 881,240 6mA sites accounting for ~0.051% of the total adenines. [G/C]AGG[C/T] was the most significantly associated motif with 6mA modification. 6mA sites were enriched in the coding regions and mark actively transcribed genes in human cells. DNA 6mA and N6-demethyladenine modification in the human genome were mediated by methyltransferase N6AMT1 and demethylase ALKBH1, respectively. The abundance of 6mA was significantly lower in cancers, accompanied by decreased N6AMT1 and increased ALKBH1 levels, and downregulation of 6mA modification levels promoted tumorigenesis. Collectively, our results demonstrate that DNA 6mA modification is extensively present in human cells and the decrease of genomic DNA 6mA promotes human tumorigenesis. Copyright © 2018 Elsevier Inc. All rights reserved.


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