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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  |  

Characterization of four C1q/TNF-related proteins (CTRPs) from red-lip mullet (Liza haematocheila) and their transcriptional modulation in response to bacterial and pathogen-associated molecular pattern stimuli.

The structural and evolutionary linkage between tumor necrosis factor (TNF) and the globular C1q (gC1q) domain defines the C1q and TNF-related proteins (CTRPs), which are involved in diverse functions such as immune defense, inflammation, apoptosis, autoimmunity, and cell differentiation. In this study, red-lip mullet (Liza haematocheila) CTRP4-like (MuCTRP4-like), CTRP5 (MuCTRP5), CTRP6 (MuCTRP6), and CTRP7 (MuCTRP7) were identified from the red-lip mullet transcriptome database and molecularly characterized. According to in silico analysis, coding sequences of MuCTRP4-like, MuCTRP5, MuCTRP6, and MuCTRP7 consisted of 1128, 753, 729, and 888 bp open reading frames (ORF), respectively and encoded 375, 250, 242, and 295 amino acids, respectively. All CTRPs possessed a putative C1q domain. Additionally, MuCTRP5, MuCTRP6, and MuCTRP7 consisted of a collagen region. Phylogenetic analysis exemplified that MuCTRPs were distinctly clustered with the respective CTRP orthologs. Tissue-specific expression analysis demonstrated that MuCTRP4-like was mostly expressed in the blood and intestine. Moreover, MuCTRP6 was highly expressed in the blood, whereas MuCTRP5 and MuCTRP7 were predominantly expressed in the muscle and stomach, respectively. According to the temporal expression in blood, all MuCTRPs exhibited significant modulations in response to polyinosinic:polycytidylic acid (poly I:C) and Lactococcus garvieae (L. garvieae). MuCTRP4-like, MuCTRP5, and MuCTRP6 showed significant upregulation in response to lipopolysaccharides (LPS). The results of this study suggest the potential involvement of Mullet CTRPs in post-immune responses. Copyright © 2018. Published by Elsevier Ltd.


September 22, 2019  |  

Robust and effective methodologies for cryopreservation and DNA extraction from anaerobic gut fungi.

Cell storage and DNA isolation are essential to developing an expanded suite of microorganisms for biotechnology. However, many features of non-model microbes, such as an anaerobic lifestyle and rigid cell wall, present formidable challenges to creating strain repositories and extracting high quality genomic DNA. Here, we establish accessible, high efficiency, and robust techniques to store lignocellulolytic anaerobic gut fungi long term without specialized equipment. Using glycerol as a cryoprotectant, gut fungal isolates were preserved for a minimum of 23 months at -80 °C. Unlike previously reported approaches, this improved protocol is non-toxic and rapid, with samples surviving twice as long with negligible growth impact. Genomic DNA extraction for these isolates was optimized to yield samples compatible with next generation sequencing platforms (e.g. Illumina, PacBio). Popular DNA isolation kits and precipitation protocols yielded preps that were unsuitable for sequencing due to carbohydrate contaminants from the chitin-rich cell wall and extensive energy reserves of gut fungi. To address this, we identified a proprietary method optimized for hardy plant samples that rapidly yielded DNA fragments in excess of 10 kb with minimal RNA, protein or carbohydrate contamination. Collectively, these techniques serve as fundamental tools to manipulate powerful biomass-degrading gut fungi and improve their accessibility among researchers. Copyright © 2015 Elsevier Ltd. All rights reserved.


September 22, 2019  |  

Researches on transcriptome sequencing in the study of traditional Chinese medicine

Due to its incomparable advantages, the application of transcriptome sequencing in the study of traditional Chinese medicine attracts more and more attention of researchers, which greatly promote the development of traditional Chinese medicine. In this paper, the applications of transcriptome sequencing in traditional Chinese medicine were summarized by reviewing recent related papers.


September 22, 2019  |  

Identification and characterization of a carboxypeptidase N1 from red lip mullet (Liza haematocheila); revealing its immune relevance.

Complement system orchestrates the innate and adaptive immunity via the activation, recruitment, and regulation of immune molecules to destroy pathogens. However, regulation of the complement is essential to avoid injuries to the autologous tissues. The present study unveils the characteristic features of an important complement component, anaphylatoxin inactivator from red lip mullet at its molecular and functional level. Mullet carboxypeptidase N1 (MuCPN1) cDNA sequence possessed an open reading frame of 1347 bp, which encoded a protein of 449 amino acids with a predicted molecular weight of 51?kDa. In silico analysis discovered two domains of PM14-Zn carboxypeptidase and a C-terminal domain of M14 N/E carboxypeptidase, two zinc-binding signature motifs, and an N-glycosylation site in the MuCPN1 sequence. Homology analysis revealed that most of the residues in the sequence are conserved among the other selected homologs. Phylogeny analysis showed that MuCPN1 closely cladded with the Maylandia zebra CPN1 and clustered together with the teleostean counterparts. A challenge experiment showed modulated expression of MuCPN1 upon polyinosinic:polycytidylic acid and Lactococcus garviae in head kidney, spleen, gill, and liver tissues. The highest upregulation of MuCPN1 was observed 24?h post infection against poly I:C in each tissue. Moreover, the highest relative expressions upon L. garviae challenge were observed at 24?h post infection in head kidney tissue and 48?h post infection in spleen, gill, and liver tissues. MuCPN1 transfected cells triggered a 2.2-fold increase of nitric oxide (NO) production upon LPS stimulation compared to the un-transfected controls suggesting that MuCPN1 is an active protease which releases arginine from complement C3a, C4a, and C5a. These results have driven certain way towards enhancing the understanding of immune role of MuCPN1 in the complement defense mechanism of red lip mullet. Copyright © 2018 Elsevier Ltd. All rights reserved.


September 22, 2019  |  

Limited effects of variable-retention harvesting on fungal communities decomposing fine roots in coastal temperate rainforests.

Fine root litter is the principal source of carbon stored in forest soils and a dominant source of carbon for fungal decomposers. Differences in decomposer capacity between fungal species may be important determinants of fine-root decomposition rates. Variable-retention harvesting (VRH) provides refuge for ectomycorrhizal fungi, but its influence on fine-root decomposers is unknown, as are the effects of functional shifts in these fungal communities on carbon cycling. We compared fungal communities decomposing fine roots (in litter bags) under VRH, clear-cut, and uncut stands at two sites (6 and 13 years postharvest) and two decay stages (43 days and 1 year after burial) in Douglas fir forests in coastal British Columbia, Canada. Fungal species and guilds were identified from decomposed fine roots using high-throughput sequencing. Variable retention had short-term effects on ß-diversity; harvest treatment modified the fungal community composition at the 6-year-postharvest site, but not at the 13-year-postharvest site. Ericoid and ectomycorrhizal guilds were not more abundant under VRH, but stand age significantly structured species composition. Guild composition varied by decay stage, with ruderal species later replaced by saprotrophs and ectomycorrhizae. Ectomycorrhizal abundance on decomposing fine roots may partially explain why fine roots typically decompose more slowly than surface litter. Our results indicate that stand age structures fine-root decomposers but that decay stage is more important in structuring the fungal community than shifts caused by harvesting. The rapid postharvest recovery of fungal communities decomposing fine roots suggests resiliency within this community, at least in these young regenerating stands in coastal British Columbia.IMPORTANCE Globally, fine roots are a dominant source of carbon in forest soils, yet the fungi that decompose this material and that drive the sequestration or respiration of this carbon remain largely uncharacterized. Fungi vary in their capacity to decompose plant litter, suggesting that fungal community composition is an important determinant of decomposition rates. Variable-retention harvesting is a forestry practice that modifies fungal communities by providing refuge for ectomycorrhizal fungi. We evaluated the effects of variable retention and clear-cut harvesting on fungal communities decomposing fine roots at two sites (6 and 13 years postharvest), at two decay stages (43 days and 1 year), and in uncut stands in temperate rainforests. Harvesting impacts on fungal community composition were detected only after 6 years after harvest. We suggest that fungal community composition may be an important factor that reduces fine-root decomposition rates relative to those of above-ground plant litter, which has important consequences for forest carbon cycling. Copyright © 2018 American Society for Microbiology.


September 22, 2019  |  

Capturing natural product biosynthetic pathways from uncultivated symbiotic bacteria of marine sponges through metagenome mining: a mini-review

Symbiotic bacteria associated with marine sponges have frequently been proposed as the true producer of many bioactive natural products with potent anticancer activities. However, the majority of these complex symbiotic bacteria cannot be cultivated under laboratory conditions, hampering efforts to access and develop their potent compounds for therapeutic applications. Metagenome mining is a powerful cultivation-independent tool that can be used to search for new natural product biosynthetic pathways from highly complex bacterial consortia. Some notable examples of natural products, in which their biosynthetic pathways have been cloned by metagenome mining are onnamide A, psymberin, polytheonamides, calyculin, and misakinolide A. Subsequent expression of the pathways in easily culturable bacteria, such as Escherichia coli, could lead to the sustainable production of rare promising natural products. This review discusses principles of metagenome mining developed to gain access to natural product biosynthetic pathways from uncultured symbiotic bacteria of marine sponges. This includes detecting biosynthetic genes in sponge metagenome, creating large metagenomic library, rapid screening of metagenomic library, and clone sequencing. For many natural products made by modular polyketide synthases (PKSs) and hybrids with non-ribosomal peptide synthetases (NRPSs), their biosynthetic pathways as well as structures of final products can be predicted with high accuracy through bioinformatic analysis and sometimes combined with functional proof. Further metagenome sequencing integrated with single-cell analysis and chemical studies could provide insights into the remarkable biosynthetic capacity of uncultivated bacterial symbionts, thereby facilitating the discovery and sustainable production of a wide diversity of sponge-derived complex compounds.


September 22, 2019  |  

Molecular genetic diversity and characterization of conjugation genes in the fish parasite Ichthyophthirius multifiliis.

Ichthyophthirius multifiliis is the etiologic agent of “white spot”, a commercially important disease of freshwater fish. As a parasitic ciliate, I. multifiliis infects numerous host species across a broad geographic range. Although Ichthyophthirius outbreaks are difficult to control, recent sequencing of the I. multifiliis genome has revealed a number of potential metabolic pathways for therapeutic intervention, along with likely vaccine targets for disease prevention. Nonetheless, major gaps exist in our understanding of both the life cycle and population structure of I. multifiliis in the wild. For example, conjugation has never been described in this species, and it is unclear whether I. multifiliis undergoes sexual reproduction, despite the presence of a germline micronucleus. In addition, no good methods exist to distinguish strains, leaving phylogenetic relationships between geographic isolates completely unresolved. Here, we compared nucleotide sequences of SSUrDNA, mitochondrial NADH dehydrogenase subunit I and cox-1 genes, and 14 somatic SNP sites from nine I. multifiliis isolates obtained from four different states in the US since 1995. The mitochondrial sequences effectively distinguished the isolates from one another and divided them into at least two genetically distinct groups. Furthermore, none of the nine isolates shared the same composition of the 14 somatic SNP sites, suggesting that I. multifiliis undergoes sexual reproduction at some point in its life cycle. Finally, compared to the well-studied free-living ciliates Tetrahymena thermophila and Paramecium tetraurelia, I. multifiliis has lost 38% and 29%, respectively, of 16 experimentally confirmed conjugation-related genes, indicating that mechanistic differences in sexual reproduction are likely to exist between I. multifiliis and other ciliate species. Copyright © 2015 Elsevier Inc. All rights reserved.


September 22, 2019  |  

The influence of energy harvesting strategies on performance and microbial community for sediment microbial fuel cells

Sediment microbial fuel cells (SMFCs) are being developed as potential energy sources where remote sensing and monitoring would be useful. Several energy harvesting techniques for SMFCs have emerged, but effects of these different strategies on startup, performance, and microbial community are not well understood. We investigated these effects by comparing a continuous energy harvesting (CEH) strategy with an intermittent energy harvesting (IEH) strategy. During startup, IEH systems immediately produced higher power and were cathode limited. CEH systems exhibited a gradual power increase and were anode-limited during startup. Both system types produced similar amounts of steady-state power, 1.5 mW ft-2 (16 mW m-2) when optimized. However, an IEH system using unoptimized settings could not be subsequently switched to optimal settings and produce expected power levels. The choice of energy harvester did not appear to significantly affect steady-state community structure. Anodes were dominated by ?- and d-proteobacteria while a- and ?-proteobacteria dominated cathodes. The results suggest performance and community structure are unaffected by energy harvesting strategy, but that startup conditions influence the initial amount of harvested energy and steady-state performance, suggesting future investigations into optimizing startup of these systems are critical for rapidly generating maximum power.


September 22, 2019  |  

Transcriptome profiling using single-molecule direct RNA sequencing approach for in-depth understanding of genes in secondary metabolism pathways of Camellia sinensis.

Characteristic secondary metabolites, including flavonoids, theanine and caffeine, are important components of Camellia sinensis, and their biosynthesis has attracted widespread interest. Previous studies on the biosynthesis of these major secondary metabolites using next-generation sequencing technologies limited the accurately prediction of full-length (FL) splice isoforms. Herein, we applied single-molecule sequencing to pooled tea plant tissues, to provide a more complete transcriptome of C. sinensis. Moreover, we identified 94 FL transcripts and four alternative splicing events for enzyme-coding genes involved in the biosynthesis of flavonoids, theanine and caffeine. According to the comparison between long-read isoforms and assemble transcripts, we improved the quality and accuracy of genes sequenced by short-read next-generation sequencing technology. The resulting FL transcripts, together with the improved assembled transcripts and identified alternative splicing events, enhance our understanding of genes involved in the biosynthesis of characteristic secondary metabolites in C. sinensis.


September 22, 2019  |  

Candidatus Dactylopiibacterium carminicum, a nitrogen-fixing symbiont of Dactylopius cochineal insects (Hemiptera: Coccoidea: Dactylopiidae)

The domesticated carmine cochineal Dactylopius coccus (scale insect) has commercial value and has been used for more than 500?years for natural red pigment production. Besides the domesticated cochineal, other wild Dactylopius species such as Dactylopius opuntiae are found in the Americas, all feeding on nutrient poor sap from native cacti. To compensate nutritional deficiencies, many insects harbor symbiotic bacteria which provide essential amino acids or vitamins to their hosts. Here, we characterized a symbiont from the carmine cochineal insects, Candidatus Dactylopiibacterium carminicum (betaproteobacterium, Rhodocyclaceae family) and found it in D. coccus and in D. opuntiae ovaries by fluorescent in situ hybridization, suggesting maternal inheritance. Bacterial genomes recovered from metagenomic data derived from whole insects or tissues both from D. coccus and from D. opuntiae were around 3.6?Mb in size. Phylogenomics showed that dactylopiibacteria constituted a closely related clade neighbor to nitrogen fixing bacteria from soil or from various plants including rice and other grass endophytes. Metabolic capabilities were inferred from genomic analyses, showing a complete operon for nitrogen fixation, biosynthesis of amino acids and vitamins and putative traits of anaerobic or microoxic metabolism as well as genes for plant interaction. Dactylopiibacterium nif gene expression and acetylene reduction activity detecting nitrogen fixation were evidenced in D. coccus hemolymph and ovaries, in congruence with the endosymbiont fluorescent in situ hybridization location. Dactylopiibacterium symbionts may compensate for the nitrogen deficiency in the cochineal diet. In addition, this symbiont may provide essential amino acids, recycle uric acid, and increase the cochineal life span.


September 22, 2019  |  

A comprehensive quality evaluation system for complex herbal medicine using PacBio sequencing, PCR-denaturing gradient gel electrophoresis, and several chemical approaches.

Herbal medicine is a major component of complementary and alternative medicine, contributing significantly to the health of many people and communities. Quality control of herbal medicine is crucial to ensure that it is safe and sound for use. Here, we investigated a comprehensive quality evaluation system for a classic herbal medicine, Danggui Buxue Formula, by applying genetic-based and analytical chemistry approaches to authenticate and evaluate the quality of its samples. For authenticity, we successfully applied two novel technologies, third-generation sequencing and PCR-DGGE (denaturing gradient gel electrophoresis), to analyze the ingredient composition of the tested samples. For quality evaluation, we used high performance liquid chromatography assays to determine the content of chemical markers to help estimate the dosage relationship between its two raw materials, plant roots of Huangqi and Danggui. A series of surveys were then conducted against several exogenous contaminations, aiming to further access the efficacy and safety of the samples. In conclusion, the quality evaluation system demonstrated here can potentially address the authenticity, quality, and safety of herbal medicines, thus providing novel insight for enhancing their overall quality control. Highlight: We established a comprehensive quality evaluation system for herbal medicine, by combining two genetic-based approaches third-generation sequencing and DGGE (denaturing gradient gel electrophoresis) with analytical chemistry approaches to achieve the authentication and quality connotation of the samples.


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.


September 22, 2019  |  

Using PacBio long-read high-throughput microbial gene amplicon sequencing to evaluate infant formula safety.

Infant formula (IF) requires a strict microbiological standard because of the high vulnerability of infants to foodborne diseases. The current study used the PacBio single molecule real-time (SMRT) sequencing platform to generate full-length 16S rRNA-based bacterial microbiota profiles of thirty Chinese domestic and imported IF samples. A total of 600 species were identified, dominated by Streptococcus thermophilus, Lactococcus lactis and Lactococcus piscium. Distinctive bacterial profiles were observed between the two sample groups, as confirmed with both principal coordinate analysis and multivariate analysis of variance. Moreover, the product whey protein nitrogen index (WPNI), representing the degree of preheating, negatively correlated with the relative abundances of the Bacillus genus. Our study has demonstrated the application of the PacBio SMRT sequencing platform in assessing the bacterial contamination of IF products, which is of interest to the dairy industry for effective monitoring of microbial quality and safety during production.


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