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Asset Tag: Third generation sequencing

September 22, 2019

Predominant contribution of cis-regulatory divergence in the evolution of mouse alternative splicing.

Divergence of alternative splicing represents one of the major driving forces to shape phenotypic diversity during evolution. However, the extent to which these divergences could be explained by the evolving cis-regulatory versus trans-acting factors remains unresolved. To globally investigate the relative contributions of the two factors for the first time in mammals, we measured splicing difference between C57BL/6J and SPRET/EiJ mouse strains and allele-specific splicing pattern in their F1 hybrid. Out of 11,818 alternative splicing events expressed in the cultured fibroblast cells, we identified 796 with significant difference between the parental strains. After integrating allele-specific data from F1 hybrid, we demonstrated that these events could be predominately attributed to cis-regulatory variants, including those residing at and beyond canonical splicing sites. Contrary to previous observations in Drosophila, such predominant contribution was consistently observed across different types of alternative splicing. Further analysis of liver tissues from the same mouse strains and reanalysis of published datasets on other strains showed similar trends, implying in general the predominant contribution of cis-regulatory changes in the evolution of mouse alternative splicing. © 2015 The Authors. Published under the terms of the CC BY 4.0 license.

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September 22, 2019

Precise fecal microbiome of the herbivorous Tibetan antelope inhabiting high-altitude alpine plateau

The metataxonomic approach combining 16S rRNA gene amplicon sequencing using the PacBio technology with the application of the operational phylogenetic unit (OPU) approach, has been used to analyze the fecal microbial composition of the high-altitude and herbivorous Tibetan antelopes. The fecal samples of the antelope were collected in Hoh Xil National Nature Reserve, at an altitude over 4500 m, the largest depopulated zone in Qinghai-Tibetan Plateau, China, where non-native animals or humans may experience life-threatening acute mountain sickness. In total, 104 antelope fecal samples were enrolled in this study, and were clustered into 61,258 operational taxonomic units (OTUs) at an identity of 98.7% and affiliated with 757 OPUs, including 144 known species, 256 potentially new species, 103 potentially higher taxa within known lineages. In addition, 254 comprised sequences not affiliating with any known family, and the closest relatives were unclassified lineages of existing orders or classes. A total of 42 out of 757 OPUs conformed to the core fecal microbiome, of which four major lineages, namely, un-cultured Ruminococcaceae, Lachnospiraceae, Akkermansia and Christensenellaceae were associated with human health or longevity. The current study reveals that the fecal core microbiome of antelope is mainly composited of uncultured bacteria. The most abundant core taxa, namely, uncultured Ruminococcaceae, uncultured Akkermansia, uncultured Bacteroides, uncultured Christensenellaceae, uncultured Mollicutes, and uncultured Lachnospiraceae, may represent new bacterial candidates at high taxa levels, and several may have beneficial roles in health promotion or anti-intestinal dysbiosis. These organisms should be further isolated and evaluated for potential effect on human health and longevity.

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September 22, 2019

Recent insights into the tick microbiome gained through next-generation sequencing.

The tick microbiome comprises communities of microorganisms, including viruses, bacteria and eukaryotes, and is being elucidated through modern molecular techniques. The advent of next-generation sequencing (NGS) technologies has enabled the genes and genomes within these microbial communities to be explored in a rapid and cost-effective manner. The advantages of using NGS to investigate microbiomes surpass the traditional non-molecular methods that are limited in their sensitivity, and conventional molecular approaches that are limited in their scalability. In recent years the number of studies using NGS to investigate the microbial diversity and composition of ticks has expanded. Here, we provide a review of NGS strategies for tick microbiome studies and discuss the recent findings from tick NGS investigations, including the bacterial diversity and composition, influential factors, and implications of the tick microbiome.

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September 22, 2019

Long reads: their purpose and place.

In recent years long-read technologies have moved from being a niche and specialist field to a point of relative maturity likely to feature frequently in the genomic landscape. Analogous to next generation sequencing, the cost of sequencing using long-read technologies has materially dropped whilst the instrument throughput continues to increase. Together these changes present the prospect of sequencing large numbers of individuals with the aim of fully characterizing genomes at high resolution. In this article, we will endeavour to present an introduction to long-read technologies showing: what long reads are; how they are distinct from short reads; why long reads are useful and how they are being used. We will highlight the recent developments in this field, and the applications and potential of these technologies in medical research, and clinical diagnostics and therapeutics.

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September 22, 2019

Soil microbial communities and elk foraging intensity: implications for soil biogeochemical cycling in the sagebrush steppe.

Foraging intensity of large herbivores may exert an indirect top-down ecological force on soil microbial communities via changes in plant litter inputs. We investigated the responses of the soil microbial community to elk (Cervus elaphus) winter range occupancy across a long-term foraging exclusion experiment in the sagebrush steppe of the North American Rocky Mountains, combining phylogenetic analysis of fungi and bacteria with shotgun metagenomics and extracellular enzyme assays. Winter foraging intensity was associated with reduced bacterial richness and increasingly distinct bacterial communities. Although fungal communities did not respond linearly to foraging intensity, a greater ß-diversity response to winter foraging exclusion was observed. Furthermore, winter foraging exclusion increased soil cellulolytic and hemicellulolytic enzyme potential and higher foraging intensity reduced chitinolytic gene abundance. Thus, future changes in winter range occupancy may shape biogeochemical processes via shifts in microbial communities and subsequent changes to their physiological capacities to cycle soil C and N.© 2017 John Wiley & Sons Ltd/CNRS.

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September 22, 2019

Full-length RNA sequencing reveals unique transcriptome composition in bermudagrass.

Bermudagrass [Cynodon dactylon (L.) Pers.] is an important perennial warm-season turfgrass species with great economic value. However, the reference genome and transcriptome information are still deficient in bermudagrass, which severely impedes functional and molecular breeding studies. In this study, through analyzing a mixture sample of leaves, stolons, shoots, roots and flowers with single-molecule long-read sequencing technology from Pacific Biosciences (PacBio), we reported the first full-length transcriptome dataset of bermudagrass (C. dactylon cultivar Yangjiang) comprising 78,192 unigenes. Among the unigenes, 66,409 were functionally annotated, whereas 27,946 were found to have two or more isoforms. The annotated full-length unigenes provided many new insights into gene sequence characteristics and systematic phylogeny of bermudagrass. By comparison with transcriptome dataset in nine grass species, KEGG pathway analyses further revealed that C4 photosynthesis-related genes, notably the phosphoenolpyruvate carboxylase and pyruvate, phosphate dikinase genes, are specifically enriched in bermudagrass. These results not only explained the possible reason why bermudagrass flourishes in warm areas but also provided a solid basis for future studies in this important turfgrass species. Copyright © 2018 Elsevier Masson SAS. All rights reserved.

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September 22, 2019

Evaluation of 16S rRNA amplicon sequencing using two next-generation sequencing technologies for phylogenetic analysis of the rumen bacterial community in steers.

Next generation sequencing technologies have vastly changed the approach of sequencing of the 16S rRNA gene for studies in microbial ecology. Three distinct technologies are available for large-scale 16S sequencing. All three are subject to biases introduced by sequencing error rates, amplification primer selection, and read length, which can affect the apparent microbial community. In this study, we compared short read 16S rRNA variable regions, V1-V3, with that of near-full length 16S regions, V1-V8, using highly diverse steer rumen microbial communities, in order to examine the impact of technology selection on phylogenetic profiles. Short paired-end reads from the Illumina MiSeq platform were used to generate V1-V3 sequence, while long “circular consensus” reads from the Pacific Biosciences RSII instrument were used to generate V1-V8 data. The two platforms revealed similar microbial operational taxonomic units (OTUs), as well as similar species richness, Good’s coverage, and Shannon diversity metrics. However, the V1-V8 amplified ruminal community resulted in significant increases in several orders of taxa, such as phyla Proteobacteria and Verrucomicrobia (P < 0.05). Taxonomic classification accuracy was also greater in the near full-length read. UniFrac distance matrices using jackknifed UPGMA clustering also noted differences between the communities. These data support the consensus that longer reads result in a finer phylogenetic resolution that may not be achieved by shorter 16S rRNA gene fragments. Our work on the cattle rumen bacterial community demonstrates that utilizing near full-length 16S reads may be useful in conducting a more thorough study, or for developing a niche-specific database to use in analyzing data from shorter read technologies when budgetary constraints preclude use of near-full length 16S sequencing. Copyright © 2016 Elsevier B.V. All rights reserved.

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September 22, 2019

Divergent brain gene expression profiles between alternative behavioural helper types in a cooperative breeder.

Juveniles of the cooperatively breeding cichlid fish Neolamprologus pulcher either consistently provide help in form of alloparental egg care (“cleaners”) or consistently abstain from helping (“noncleaners”). These phenotypes are not based on heritable genetic differences. Instead, they arise during ontogeny, which should lead to differences in brain structure or physiology, a currently untested prediction. We compared brain gene expression profiles of cleaners and noncleaners in two experimental conditions, a helping opportunity and a control condition. We aimed to identify (a) expression differences between cleaners and noncleaners in the control, (b) changes in gene expression induced by the opportunity and (c) differences in plasticity of gene expression between cleaners and noncleaners. Control cleaners and noncleaners differed in the expression of a single gene, irx2, which regulates neural differentiation. During the opportunity, cleaners and noncleaners had three upregulated genes in common, which were implicated in neuroplasticity, hormonal signalling and cell proliferation. Thus, the stimulus in the opportunity was sufficiently salient. Cleaners also showed higher expression of seven additional genes that were unique to the opportunity. One of these cleaner-specific genes is implicated in neuropeptide metabolism, indicating that this process is associated with cleaning performance. This suggests that the two types employed different pathways to integrate social information, preparing them for accelerated reaction to future opportunities. Interestingly, three developmental genes were downregulated between the control and the opportunity in cleaners only. Our results indicate that the two behavioural types responded differently to the helping opportunity and that only cleaners responded by downregulating developmental genes.© 2018 John Wiley & Sons Ltd.

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September 22, 2019

Identification of a novel fusion transcript between human relaxin-1 (RLN1) and human relaxin-2 (RLN2) in prostate cancer.

Simultaneous expression of highly homologous RLN1 and RLN2 genes in prostate impairs their accurate delineation. We used PacBio SMRT sequencing and RNA-Seq in LNCaP cells in order to dissect the expression of RLN1 and RLN2 variants. We identified a novel fusion transcript comprising the RLN1 and RLN2 genes and found evidence of its expression in the normal and prostate cancer tissues. The RLN1-RLN2 fusion putatively encodes RLN2 isoform with the deleted secretory signal peptide. The identification of the fusion transcript provided information to determine unique RLN1-RLN2 fusion and RLN1 regions. The RLN1-RLN2 fusion was co-expressed with RLN1 in LNCaP cells, but the two gene products were inversely regulated by androgens. We showed that RLN1 is underrepresented in common PCa cell lines in comparison to normal and PCa tissue. The current study brings a highly relevant update to the relaxin field, and will encourage further studies of RLN1 and RLN2 in PCa and broader. Copyright © 2015 The Authors. Published by Elsevier Ireland Ltd.. All rights reserved.

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September 22, 2019

The gut commensal microbiome of Drosophila melanogaster is modified by the endosymbiont Wolbachia.

Endosymbiotic Wolbachia bacteria and the gut microbiome have independently been shown to affect several aspects of insect biology, including reproduction, development, life span, stem cell activity, and resistance to human pathogens, in insect vectors. This work shows that Wolbachia bacteria, which reside mainly in the fly germline, affect the microbial species present in the fly gut in a lab-reared strain. Drosophila melanogaster hosts two main genera of commensal bacteria-Acetobacter and Lactobacillus. Wolbachia-infected flies have significantly reduced titers of Acetobacter. Sampling of the microbiome of axenic flies fed with equal proportions of both bacteria shows that the presence of Wolbachia bacteria is a significant determinant of the composition of the microbiome throughout fly development. However, this effect is host genotype dependent. To investigate the mechanism of microbiome modulation, the effect of Wolbachia bacteria on Imd and reactive oxygen species pathways, the main regulators of immune response in the fly gut, was measured. The presence of Wolbachia bacteria does not induce significant changes in the expression of the genes for the effector molecules in either pathway. Furthermore, microbiome modulation is not due to direct interaction between Wolbachia bacteria and gut microbes. Confocal analysis shows that Wolbachia bacteria are absent from the gut lumen. These results indicate that the mechanistic basis of the modulation of composition of the microbiome by Wolbachia bacteria is more complex than a direct bacterial interaction or the effect of Wolbachia bacteria on fly immunity. The findings reported here highlight the importance of considering the composition of the gut microbiome and host genetic background during Wolbachia-induced phenotypic studies and when formulating microbe-based disease vector control strategies. IMPORTANCE Wolbachia bacteria are intracellular bacteria present in the microbiome of a large fraction of insects and parasitic nematodes. They can block mosquitos’ ability to transmit several infectious disease-causing pathogens, including Zika, dengue, chikungunya, and West Nile viruses and malaria parasites. Certain extracellular bacteria present in the gut lumen of these insects can also block pathogen transmission. However, our understanding of interactions between Wolbachia and gut bacteria and how they influence each other is limited. Here we show that the presence of Wolbachia strain wMel changes the composition of gut commensal bacteria in the fruit fly. Our findings implicate interactions between bacterial species as a key factor in determining the overall composition of the microbiome and thus reveal new paradigms to consider in the development of disease control strategies.

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September 22, 2019

Improved metagenome assemblies and taxonomic binning using long-read circular consensus sequence data.

DNA assembly is a core methodological step in metagenomic pipelines used to study the structure and function within microbial communities. Here we investigate the utility of Pacific Biosciences long and high accuracy circular consensus sequencing (CCS) reads for metagenomic projects. We compared the application and performance of both PacBio CCS and Illumina HiSeq data with assembly and taxonomic binning algorithms using metagenomic samples representing a complex microbial community. Eight SMRT cells produced approximately 94 Mb of CCS reads from a biogas reactor microbiome sample that averaged 1319 nt in length and 99.7% accuracy. CCS data assembly generated a comparative number of large contigs greater than 1?kb, to those assembled from a ~190x larger HiSeq dataset (~18 Gb) produced from the same sample (i.e approximately 62% of total contigs). Hybrid assemblies using PacBio CCS and HiSeq contigs produced improvements in assembly statistics, including an increase in the average contig length and number of large contigs. The incorporation of CCS data produced significant enhancements in taxonomic binning and genome reconstruction of two dominant phylotypes, which assembled and binned poorly using HiSeq data alone. Collectively these results illustrate the value of PacBio CCS reads in certain metagenomics applications.

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September 22, 2019

Unveiling the complexity of the maize transcriptome by single-molecule long-read sequencing.

Zea mays is an important genetic model for elucidating transcriptional networks. Uncertainties about the complete structure of mRNA transcripts limit the progress of research in this system. Here, using single-molecule sequencing technology, we produce 111,151 transcripts from 6 tissues capturing ~70% of the genes annotated in maize RefGen_v3 genome. A large proportion of transcripts (57%) represent novel, sometimes tissue-specific, isoforms of known genes and 3% correspond to novel gene loci. In other cases, the identified transcripts have improved existing gene models. Averaging across all six tissues, 90% of the splice junctions are supported by short reads from matched tissues. In addition, we identified a large number of novel long non-coding RNAs and fusion transcripts and found that DNA methylation plays an important role in generating various isoforms. Our results show that characterization of the maize B73 transcriptome is far from complete, and that maize gene expression is more complex than previously thought.

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September 22, 2019

Multiple regulatory networks are activated during cold stress in Medicago sativa L.

Cultivated alfalfa (Medicago sativa L.) is one of the most important perennial legume forages in the world, and it has considerable potential as a valuable forage crop for livestock. However, the molecular mechanisms underlying alfalfa responses to cold stress are largely unknown. In this study, the transcriptome changes in alfalfa under cold stress at 4 °C for 2, 6, 24, and 48 h (three replicates for each time point) were analyzed using the high-throughput sequencing platform, BGISEQ-500, resulting in the identification of 50,809 annotated unigenes and 5283 differentially expressed genes (DEGs). Metabolic pathway enrichment analysis demonstrated that the DEGs were involved in carbohydrate metabolism, photosynthesis, plant hormone signal transduction, and the biosynthesis of amino acids. Moreover, the physiological changes of glutathione and proline content, catalase, and peroxidase activity were in accordance with dynamic transcript profiles of the relevant genes. Additionally, some transcription factors might play important roles in the alfalfa response to cold stress, as determined by the expression pattern of the related genes during 48 h of cold stress treatment. These findings provide valuable information for identifying and characterizing important components in the cold signaling network in alfalfa and enhancing the understanding of the molecular mechanisms underlying alfalfa responses to cold stress.

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September 22, 2019

ISOdb: A comprehensive database of full-length isoforms generated by Iso-Seq.

The accurate landscape of transcript isoforms plays an important role in the understanding of gene function and gene regulation. However, building complete transcripts is very challenging for short reads generated using next-generation sequencing. Fortunately, isoform sequencing (Iso-Seq) using single-molecule sequencing technologies, such as PacBio SMRT, provides long reads spanning entire transcript isoforms which do not require assembly. Therefore, we have developed ISOdb, a comprehensive resource database for hosting and carrying out an in-depth analysis of Iso-Seq datasets and visualising the full-length transcript isoforms. The current version of ISOdb has collected 93 publicly available Iso-Seq samples from eight species and presents the samples in two levels: (1) sample level, including metainformation, long read distribution, isoform numbers, and alternative splicing (AS) events of each sample; (2) gene level, including the total isoforms, novel isoform number, novel AS number, and isoform visualisation of each gene. In addition, ISOdb provides a user interface in the website for uploading sample information to facilitate the collection and analysis of researchers’ datasets. Currently, ISOdb is the first repository that offers comprehensive resources and convenient public access for hosting, analysing, and visualising Iso-Seq data, which is freely available.

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