September 22, 2019  |  

Uncovering full-length transcript isoforms of sugarcane cultivar Khon Kaen 3 using single-molecule long-read sequencing.

Sugarcane is an important global food crop and energy resource. To facilitate the sugarcane improvement program, genome and gene information are important for studying traits at the molecular level. Most currently available transcriptome data for sugarcane were generated using second-generation sequencing platforms, which provide short reads. The de novo assembled transcripts from these data are limited in length, and hence may be incomplete and inaccurate, especially for long RNAs.We generated a transcriptome dataset of leaf tissue from a commercial Thai sugarcane cultivar Khon Kaen 3 (KK3) using PacBio RS II single-molecule long-read sequencing by the Iso-Seq method. Short-read RNA-Seq data were generated from the same RNA sample using the Ion Proton platform for reducing base calling errors.A total of 119,339 error-corrected transcripts were generated with the N50 length of 3,611 bp, which is on average longer than any previously reported sugarcane transcriptome dataset. 110,253 sequences (92.4%) contain an open reading frame (ORF) of at least 300 bp long with ORF N50 of 1,416 bp. The mean lengths of 5′ and 3′ untranslated regions in 73,795 sequences with complete ORFs are 1,249 and 1,187 bp, respectively. 4,774 transcripts are putatively novel full-length transcripts which do not match with a previous Iso-Seq study of sugarcane. We annotated the functions of 68,962 putative full-length transcripts with at least 90% coverage when compared with homologous protein coding sequences in other plants.The new catalog of transcripts will be useful for genome annotation, identification of splicing variants, SNP identification, and other research pertaining to the sugarcane improvement program. The putatively novel transcripts suggest unique features of KK3, although more data from different tissues and stages of development are needed to establish a reference transcriptome of this cultivar.


September 22, 2019  |  

SQANTI: extensive characterization of long-read transcript sequences for quality control in full-length transcriptome identification and quantification.

High-throughput sequencing of full-length transcripts using long reads has paved the way for the discovery of thousands of novel transcripts, even in well-annotated mammalian species. The advances in sequencing technology have created a need for studies and tools that can characterize these novel variants. Here, we present SQANTI, an automated pipeline for the classification of long-read transcripts that can assess the quality of data and the preprocessing pipeline using 47 unique descriptors. We apply SQANTI to a neuronal mouse transcriptome using Pacific Biosciences (PacBio) long reads and illustrate how the tool is effective in characterizing and describing the composition of the full-length transcriptome. We perform extensive evaluation of ToFU PacBio transcripts by PCR to reveal that an important number of the novel transcripts are technical artifacts of the sequencing approach and that SQANTI quality descriptors can be used to engineer a filtering strategy to remove them. Most novel transcripts in this curated transcriptome are novel combinations of existing splice sites, resulting more frequently in novel ORFs than novel UTRs, and are enriched in both general metabolic and neural-specific functions. We show that these new transcripts have a major impact in the correct quantification of transcript levels by state-of-the-art short-read-based quantification algorithms. By comparing our iso-transcriptome with public proteomics databases, we find that alternative isoforms are elusive to proteogenomics detection. SQANTI allows the user to maximize the analytical outcome of long-read technologies by providing the tools to deliver quality-evaluated and curated full-length transcriptomes.© 2018 Tardaguila et al.; Published by Cold Spring Harbor Laboratory Press.


September 22, 2019  |  

Hybrid sequencing of full-length cDNA transcripts of stems and leaves in Dendrobium officinale.

Dendrobium officinale is an extremely valuable orchid used in traditional Chinese medicine, so sought after that it has a higher market value than gold. Although the expression profiles of some genes involved in the polysaccharide synthesis have previously been investigated, little research has been carried out on their alternatively spliced isoforms in D. officinale. In addition, information regarding the translocation of sugars from leaves to stems in D. officinale also remains limited. We analyzed the polysaccharide content of D. officinale leaves and stems, and completed in-depth transcriptome sequencing of these two diverse tissue types using second-generation sequencing (SGS) and single-molecule real-time (SMRT) sequencing technology. The results of this study yielded a digital inventory of gene and mRNA isoform expressions. A comparative analysis of both transcriptomes uncovered a total of 1414 differentially expressed genes, including 844 that were up-regulated and 570 that were down-regulated in stems. Of these genes, one sugars will eventually be exported transporter (SWEET) and one sucrose transporter (SUT) are expressed to a greater extent in D. officinale stems than in leaves. Two glycosyltransferase (GT) and four cellulose synthase (Ces) genes undergo a distinct degree of alternative splicing. In the stems, the content of polysaccharides is twice as much as that in the leaves. The differentially expressed GT and transcription factor (TF) genes will be the focus of further study. The genes DoSWEET4 and DoSUT1 are significantly expressed in the stem, and are likely to be involved in sugar loading in the phloem.


September 22, 2019  |  

A survey of the complex transcriptome from the highly polyploid sugarcane genome using full-length isoform sequencing and de novo assembly from short read sequencing.

Despite the economic importance of sugarcane in sugar and bioenergy production, there is not yet a reference genome available. Most of the sugarcane transcriptomic studies have been based on Saccharum officinarum gene indices (SoGI), expressed sequence tags (ESTs) and de novo assembled transcript contigs from short-reads; hence knowledge of the sugarcane transcriptome is limited in relation to transcript length and number of transcript isoforms.The sugarcane transcriptome was sequenced using PacBio isoform sequencing (Iso-Seq) of a pooled RNA sample derived from leaf, internode and root tissues, of different developmental stages, from 22 varieties, to explore the potential for capturing full-length transcript isoforms. A total of 107,598 unique transcript isoforms were obtained, representing about 71% of the total number of predicted sugarcane genes. The majority of this dataset (92%) matched the plant protein database, while just over 2% was novel transcripts, and over 2% was putative long non-coding RNAs. About 56% and 23% of total sequences were annotated against the gene ontology and KEGG pathway databases, respectively. Comparison with de novo contigs from Illumina RNA-Sequencing (RNA-Seq) of the internode samples from the same experiment and public databases showed that the Iso-Seq method recovered more full-length transcript isoforms, had a higher N50 and average length of largest 1,000 proteins; whereas a greater representation of the gene content and RNA diversity was captured in RNA-Seq. Only 62% of PacBio transcript isoforms matched 67% of de novo contigs, while the non-matched proportions were attributed to the inclusion of leaf/root tissues and the normalization in PacBio, and the representation of more gene content and RNA classes in the de novo assembly, respectively. About 69% of PacBio transcript isoforms and 41% of de novo contigs aligned with the sorghum genome, indicating the high conservation of orthologs in the genic regions of the two genomes.The transcriptome dataset should contribute to improved sugarcane gene models and sugarcane protein predictions; and will serve as a reference database for analysis of transcript expression in sugarcane.


September 22, 2019  |  

Altered expression of the FMR1 splicing variants landscape in premutation carriers.

FMR1 premutation carriers (55-200 CGG repeats) are at risk for developing Fragile X-associated Tremor/Ataxia Syndrome (FXTAS), an adult onset neurodegenerative disorder. Approximately 20% of female carriers will develop Fragile X-associated Primary Ovarian Insufficiency (FXPOI), in addition to a number of clinical problems affecting premutation carriers throughout their life span. Marked elevation in FMR1 mRNA levels have been observed with premutation alleles resulting in RNA toxicity, the leading molecular mechanism proposed for the FMR1 associated disorders observed in premutation carriers. The FMR1 gene undergoes alternative splicing and we have recently reported that the relative abundance of all FMR1 mRNA isoforms is significantly increased in premutation carriers. In this study, we characterized the transcriptional FMR1 isoforms distribution pattern in different tissues and identified a total of 49 isoforms, some of which observed only in premutation carriers and which might play a role in the pathogenesis of FXTAS. Further, we investigated the distribution pattern and expression levels of the FMR1 isoforms in asymptomatic premutation carriers and in those with FXTAS and found no significant differences between the two groups. Our findings suggest that the characterization of the expression levels of the different FMR1 isoforms is fundamental for understanding the regulation of the FMR1 gene as imbalance in their expression could lead to an altered functional diversity with neurotoxic consequences. Their characterization will also help to elucidating the mechanism(s) by which “toxic gain of function” of the FMR1 mRNA may play a role in FXTAS and/or in the other FMR1-associated conditions. Copyright © 2017. Published by Elsevier B.V.


September 22, 2019  |  

Full-length transcriptome sequences and the identification of putative genes for flavonoid biosynthesis in safflower.

The flower of the safflower (Carthamus tinctorius L.) has been widely used in traditional Chinese medicine for the ability to improve cerebral blood flow. Flavonoids are the primary bioactive components in safflower, and their biosynthesis has attracted widespread interest. Previous studies mostly used second-generation sequencing platforms to survey the putative flavonoid biosynthesis genes. For a better understanding of transcription data and the putative genes involved in flavonoid biosynthesis in safflower, we carry our study.High-quality RNA was extracted from six types of safflower tissue. The RNAs of different tissues were mixed equally and used for multiple size-fractionated libraries (1-2, 2-3 and 3-6 k) library construction. Five cells were carried (2 cells for 1-2 and for 2-3 k libraries and 1 cell for 3-6 k libraries). 10.43Gb clean data and 38,302 de-redundant sequences were captured. 44 unique isoforms were annotated as encoding enzymes involved in flavonoid biosynthesis. The full length flavonoid genes were characterized and their evolutional relationship and expressional pattern were analyzed. They can be divided into eight families, with a large differences in the tissue expression. The temporal expressions under MeJA treatment were also measured, 9 genes are significantly up-regulated and 2 genes are significantly down-regulated. The genes involved in flavonoid synthesis in safflower were predicted in our study. Besides, the SSR and lncRNA are also analyzed in our study.Full-length transcriptome sequences were used in our study. The genes involved in flavonoid synthesis in safflower were predicted in our study. Combined the determination of flavonoids, CtC4H2, CtCHS3, CtCHI3, CtF3H3, CtF3H1 are mainly participated in MeJA promoting the synthesis of flavonoids. Our results also provide a valuable resource for further study on safflower.


September 22, 2019  |  

Single molecule, full-length transcript sequencing provides insight into the extreme metabolism of ruby-throated hummingbird Archilochus colubris

Hummingbirds oxidize ingested nectar sugars directly to fuel foraging but cannot sustain this fuel use during fasting periods, such as during the night or during long-distance migratory flights. Instead, fasting hummingbirds switch to oxidizing stored lipids, derived from ingested sugars. The hummingbird liver plays a key role in moderating energy homeostasis and this remarkable capacity for fuel switching. Additionally, liver is the principle location of de novo lipogenesis, which can occur at exceptionally high rates, such as during premigratory fattening. Yet understanding how this tissue and whole organism moderates energy turnover is hampered by a lack of information regarding how relevant enzymes differ in sequence, expression, and regulation. We generated a de novo transcriptome of the hummingbird liver using PacBio full-length cDNA sequencing (Iso-Seq), yielding a total of 8.6Gb of sequencing data, or 2.6M reads from 4 different size fractions. We analyzed data using the SMRTAnalysis v3.1 Iso-Seq pipeline, then clustered isoforms into gene families to generate de novo gene contigs using Cogent. We performed orthology analysis to identify closely related sequences between our transcriptome and other avian and human gene sets. Finally, we closely examined homology of critical lipid metabolism genes between our transcriptome data and avian and human genomes. We confirmed high levels of sequence divergence within hummingbird lipogenic enzymes, suggesting a high probability of adaptive divergent function in the hepatic lipogenic pathways. Our results leverage cutting-edge technology and a novel bioinformatics pipeline to provide a first direct look at the transcriptome of this incredible organism.


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  |  

Comparative genomic analysis reveals the evolution and environmental adaptation strategies of vibrios.

Vibrios are among the most diverse and ecologically important marine bacteria, which have evolved many characteristics and lifestyles to occupy various niches. The relationship between genome features and environmental adaptation strategies is an essential part for understanding the ecological functions of vibrios in the marine system. The advent of complete genome sequencing technology has provided an important method of examining the genetic characteristics of vibrios on the genomic level.Two Vibrio genomes were sequenced and found to occupy many unique orthologues families which absent from the previously genes pool of the complete genomes of vibrios. Comparative genomics analysis found vibrios encompass a steady core-genome and tremendous pan-genome with substantial gene gain and horizontal gene transfer events in the evolutionary history. Evolutionary analysis based on the core-genome tree suggested that V. fischeri emerged ~?385 million years ago, along with the occurrence of cephalopods and the flourish of fish. The relatively large genomes, the high number of 16S rRNA gene copies, and the presence of R-M systems and CRISPR system help vibrios live in various marine environments. Chitin-degrading related genes are carried in nearly all the Vibrio genomes. The number of chitinase genes in vibrios has been extremely expanded compared to which in the most recent ancestor of the genus. The chitinase A genes were estimated to have evolved along with the genus, and have undergone significant purifying selective force to conserve the ancestral state.Vibrios have experienced extremely genome expansion events during their evolutionary history, allowing them to develop various functions to spread globally. Despite their close phylogenetic relationships, vibrios were found to have a tremendous pan-genome with a steady core-genome, which indicates the highly plastic genome of the genus. Additionally, the existence of various chitin-degrading related genes and the expansion of chitinase A in the genus demonstrate the importance of the chitin utilization for vibrios. Defensive systems in the Vibrio genomes may protect them from the invasion of external DNA. These genomic features investigated here provide a better knowledge of how the evolutionary process has forged Vibrio genomes to occupy various niches.


September 22, 2019  |  

Comparative genome and methylome analysis reveals restriction/modification system diversity in the gut commensal Bifidobacterium breve.

Bifidobacterium breve represents one of the most abundant bifidobacterial species in the gastro-intestinal tract of breast-fed infants, where their presence is believed to exert beneficial effects. In the present study whole genome sequencing, employing the PacBio Single Molecule, Real-Time (SMRT) sequencing platform, combined with comparative genome analysis allowed the most extensive genetic investigation of this taxon. Our findings demonstrate that genes encoding Restriction/Modification (R/M) systems constitute a substantial part of the B. breve variable gene content (or variome). Using the methylome data generated by SMRT sequencing, combined with targeted Illumina bisulfite sequencing (BS-seq) and comparative genome analysis, we were able to detect methylation recognition motifs and assign these to identified B. breve R/M systems, where in several cases such assignments were confirmed by restriction analysis. Furthermore, we show that R/M systems typically impose a very significant barrier to genetic accessibility of B. breve strains, and that cloning of a methyltransferase-encoding gene may overcome such a barrier, thus allowing future functional investigations of members of this species.


September 22, 2019  |  

Complete genome of Cobetia marina JCM 21022T and phylogenomic analysis of the family Halomonadaceae

Cobetia marina is a model proteobacteria in researches on marine biofouling. Its taxonomic nomenclature has been revised many times over the past few decades. To better understand the role of the surface-associated lifestyle of C. marina and the phylogeny of the family Halomonadaceae, we sequenced the entire genome of C. marina JCM 21022T using single molecule real-time sequencing technology (SMRT) and performed comparative genomics and phylogenomics analyses. The circular chromosome was 4 176 300 bp with an average GC content of 62.44% and contained 3 611 predicted coding sequences, 72 tRNA genes, and 21 rRNA genes. The C. marina JCM 21022T genome contained a set of crucial genes involved in surface colonization processes. The comparative genome analysis indicated the significant diff erences between C. marina JCM 21022T and Cobetia amphilecti KMM 296 (formerly named C. marina KMM 296) resulted from sequence insertions or deletions and chromosomal recombination. Despite these diff erences, pan and core genome analysis showed similar gene functions between the two strains. The phylogenomic study of the family Halomonadaceae is reported here for the first time. We found that the relationships were well resolved among every genera tested, including Chromohalobacter, Halomonas, Cobetia, Kushneria, Zymobacter, and Halotalea.


September 22, 2019  |  

Computational Modeling of Multidrug-Resistant Bacteria

Understanding how complex phenotypes arise from individual molecules and their interactions is a primary challenge in biology, and computational approaches have been increasingly employed to tackle this task. In this chapter, we describe current efforts by FIOCRUZ and partners to develop integrated computational models of multidrug-resistant bacteria. The bacterium chosen as the main focus of this effort is Pseudomonas aeruginosa, an opportunistic pathogen associated with a broad spectrum of infections in humans. Nowadays, P. aeruginosa is one of the main problems of healthcare-associated infections (HAI) in the world, because of its great capacity of survival in hospital environments and its intrinsic resistance to many antibiotics. Our overall research objective is to use integrated computational models to accurately predict a wide range of observable cellular behaviors of multidrug-resistant P. aeruginosa CCBH4851, which is a strain belonging to the clone ST277, endemic in Brazil. In this chapter, after a brief introduction to P. aeruginosa biology, we discuss the construction of metabolic and gene regulatory networks of P. aeruginosa CCBH 4851 from its genome. We also illustrate how these networks can be integrated into a single model, and we discuss methods for identifying potential therapeutic targets through integrated models.


September 22, 2019  |  

Comparative genome analysis of jujube witches’-broom Phytoplasma, an obligate pathogen that causes jujube witches’-broom disease.

JWB phytoplasma is a kind of insect-transmitted and uncultivable bacterial plant pathogen causeing a destructive Jujube disease. To date, no genome information about JWB phytoplasma has been published, which hindered its characterization at genomic level. To understand its pathogenicity and ecology, the genome of a JWB phytoplasma isolate jwb-nky was sequenced and compared with other phytoplasmas enabled us to explore the mechanisms of genomic rearrangement.The complete genome sequence of JWB phytoplasma (jwb-nky) was determined, which consisting of one circular chromosome of 750,803 bp with a GC content of 23.3%. 694 protein-encoding genes, 2 operons for rRNA genes and 31 tRNA genes as well as 4 potential mobile units (PMUs) containing clusters of DNA repeats were identified. Based on PHIbaes analysis, a large number of genes were genome-specific and approximately 13% of JWB phytoplasma genes were predicted to be associated with virulence. Although transporters for maltose, dipeptides/oligopeptides, spermidine/putrescine, cobalt, Mn/Zn and methionine were identified, KEGG pathway analysis revealed the reduced metabolic capabilities of JWB phytoplasma. Comparative genome analyses between JWB phytoplasma and other phytoplasmas shows the occurrence of large-scale gene rearrangements. The low synteny with other phytoplasmas indicated that the expansion of multiple gene families/duplication probably occurred separately after differentiation.In this study, the complete genome sequence of a JWB phytoplasma isolate jwb-nky that causing JWB disease was reported for the first time and a number of species-specific genes were identified in the genome. The study enhanced our understandings about genomic basis and the pathogenicity mechanism of this pathogen, which will aid in the development of improved strategies for efficient management of JWB diseases.


September 22, 2019  |  

2,3-Butanediol production by the non-pathogenic bacterium Paenibacillus brasilensis.

2,3-Butanediol (2,3-BDO) is of considerable importance in the chemical, plastic, pharmaceutical, cosmetic, and food industries. The main bacterial species producing this compound are considered pathogenic, hindering large-scale productivity. The species Paenibacillus brasilensis is generally recognized as safe (GRAS) and is phylogenetically similar to P. polymyxa, a species widely used for 2,3-BDO production. Here, we demonstrate, for the first time, that P. brasilensis strains produce 2,3-BDO. Total 2,3-BDO concentrations for 15 P. brasilensis strains varied from 5.5 to 7.6 g/l after 8 h incubation at 32 °C in modified YEPD medium containing 20 g/l glucose. Strain PB24 produced 8.2 g/l of 2,3-BDO within a 12-h growth period, representing a yield of 0.43 g/g and a productivity of 0.68 g/l/h. An increase in 2,3-BDO production by strain PB24 was observed using higher concentrations of glucose, reaching 27 g/l of total 2,3-BDO in YEPD containing about 80 g/l glucose within a 72-h growth period. We sequenced the genome of P. brasilensis PB24 and uncovered at least six genes related to the 2,3-BDO pathway at four distinct loci. We also compared gene sequences related to the 2,3-BDO pathway in P. brasilensis PB24 with those of other spore-forming bacteria, and found strong similarity to P. polymyxa, P. terrae, and P. peoriae 2,3-BDO-related genes. Regulatory regions upstream of these genes indicated that they are probably co-regulated. Finally, we propose a production pathway from glucose to 2,3-BDO in P. brasilensis PB24. Although the gene encoding S-2,3-butanediol dehydrogenase (butA) was found in the genome of P. brasilensis PB24, only R,R-2,3- and meso-2,3-butanediol were detected by gas chromatography under the growth conditions tested here. Our findings can serve as a basis for further improvements to the metabolic capabilities of this little-studied Paenibacillus species in relation to production of the high-value chemical 2,3-butanediol.


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