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September 22, 2019

Ensembl 2018

The Ensembl project has been aggregating, processing, integrating and redistributing genomic datasets since the initial releases of the draft human genome, with the aim of accelerating genomics research through rapid open distribution of public data. Large amounts of raw data are thus transformed into knowledge, which is made available via a multitude of channels, in particular our browser (http://www.ensembl.org). Over time, we have expanded in multiple directions. First, our resources describe multiple fields of genomics, in particular gene annotation, comparative genomics, genetics and epigenomics. Second, we cover a growing number of genome assemblies; Ensembl Release 90 contains exactly 100. Third, our databases feed simultaneously into an array of services designed around different use cases, ranging from quick browsing to genome-wide bioinformatic analysis. We present here the latest developments of the Ensembl project, with a focus on managing an increasing number of assemblies, supporting efforts in genome interpretation and improving our browser.

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September 22, 2019

Global analysis of epigenetic regulation of gene expression in response to drought stress in Sorghum.

Abiotic stresses including drought are major limiting factors of crop yields and cause significant crop losses. Acquisition of stress tolerance to abiotic stresses requires coordinated regulation of a multitude of biochemical and physiological changes, and most of these changes depend on alterations in gene expression. The goal of this work is to perform global analysis of differential regulation of gene expression and alternative splicing, and their relationship with chromatin landscape in drought sensitive and tolerant cultivars. our Iso-Seq study revealed transcriptome-wide full-length isoforms at an unprecedented scale with over 11000 novel splice isoforms. Additionally, we uncovered alternative polyadenylation sites of ~11000 expressed genes and many novel genes. Overall, Iso-Seq results greatly enhanced sorghum gene annotations that are not only useful in analyentified differentially expressed genes and splicing events that are correlated with tzing all our RNA-seq, ChIP-seq and ATAC-seq data but also serve as a great resource to the plant biology community. Our studies idhe drought-resistant phenotype. An association between alternative splicing and chromatin accessibility was also revealed. Several computational tools developed here (TAPIS and iDiffIR) have been made freely available to the research community in analyzing alternative splicing and differential alternative splicing.

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September 22, 2019

Transcriptome profiling using Illumina- and SMRT-based RNA-seq of hot pepper for in-depth understanding of genes involved in CMV infection.

Hot pepper (Capsicum annuum L.) is becoming an increasingly important vegetable crop in the world. Cucumber mosaic virus (CMV) is a destructive virus that can cause leaf distortion and fruit lesions, affecting pepper production. However, studies on the response to CMV infection in pepper at the transcriptional level are limited. In this study, the transcript profiles of pepper leaves after CMV infection were investigated using Illumina and single-molecule real-time (SMRT) RNA-sequencing (RNA-seq). A total of 2143 differentially expressed genes (DEGs) were identified at five different stages. Gene ontology (GO) and KEGG analysis revealed that these DEGs were involved in the response to stress, defense response and plant-pathogen interaction pathways. Among these DEGs, several key genes that consistently appeared in studies of plant-pathogen interactions had increased transcript abundance after inoculation, including chitinase, pathogenesis-related (PR) protein, TMV resistance protein, WRKY transcription factor and jasmonate ZIM-domain protein. Four of these DEGs were further validated by quantitative real-time RT-PCR (qRT-PCR). Furthermore, a total of 73, 597 alternative splicing (AS) events were identified in the pepper leaves after CMV infection, distributed in 12, 615 genes. The intron retention of WRKY33 (Capana09g001251) might be involved in the regulation of CMV infection. Taken together, our study provides a transcriptome-wide insight into the molecular basis of resistance to CMV infection in pepper leaves and potential candidate genes for improving resistance cultivars. Copyright © 2018 Elsevier B.V. All rights reserved.

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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.

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September 22, 2019

Transcriptome sequencing and comparative analysis of differentially-expressed isoforms in the roots of Halogeton glomeratus under salt stress.

Although Halogeton glomeratus (H. glomeratus) has been confirmed to have a unique mechanism to regulate Na+efflux from the cytoplasm and compartmentalize Na+into leaf vacuoles, little is known about the salt tolerance mechanisms of roots under salinity stress. In the present study, transcripts were sequenced using the BGISEQ-500 sequencing platform (BGI, Wuhan, China). After quality control, approximately 24.08 million clean reads were obtained and the average mapping ratio to the reference gene was 70.00%. When comparing salt-treated samples with the control, a total of 550, 590, 1411 and 2063 DEIs were identified at 2, 6, 24 and 72h, respectively. Numerous differentially-expressed isoforms that play important roles in response and adaptation to salt condition are related to metabolic processes, cellular processes, single-organism processes, localization, biological regulation, responses to stimulus, binding, catalytic activity and transporter activity. Fifty-eight salt-induced isoforms were common to different stages of salt stress; most of these DEIs were related to signal transduction and transporters, which maybe the core isoforms regulating Na+uptake and transport in the roots of H. glomeratus. The expression patterns of 18 DEIs that were detected by quantitative real-time polymerase chain reaction were consistent with their respective changes in transcript abundance as identified by RNA-Seq technology. The present study thoroughly explored potential isoforms involved in salt tolerance on H. glomeratus roots at five time points. Our results may serve as an important resource for the H. glomeratus research community, improving our understanding of salt tolerance in halophyte survival under high salinity stress. Copyright © 2018 Elsevier B.V. All rights reserved.

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September 22, 2019

De novo transcriptome assembly of drought tolerant CAM plants, Agave deserti and Agave tequilana.

Agaves are succulent monocotyledonous plants native to xeric environments of North America. Because of their adaptations to their environment, including crassulacean acid metabolism (CAM, a water-efficient form of photosynthesis), and existing technologies for ethanol production, agaves have gained attention both as potential lignocellulosic bioenergy feedstocks and models for exploring plant responses to abiotic stress. However, the lack of comprehensive Agave sequence datasets limits the scope of investigations into the molecular-genetic basis of Agave traits.Here, we present comprehensive, high quality de novo transcriptome assemblies of two Agave species, A. tequilana and A. deserti, built from short-read RNA-seq data. Our analyses support completeness and accuracy of the de novo transcriptome assemblies, with each species having a minimum of approximately 35,000 protein-coding genes. Comparison of agave proteomes to those of additional plant species identifies biological functions of gene families displaying sequence divergence in agave species. Additionally, a focus on the transcriptomics of the A. deserti juvenile leaf confirms evolutionary conservation of monocotyledonous leaf physiology and development along the proximal-distal axis.Our work presents a comprehensive transcriptome resource for two Agave species and provides insight into their biology and physiology. These resources are a foundation for further investigation of agave biology and their improvement for bioenergy development.

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September 22, 2019

Single-cell multiomics: multiple measurements from single cells.

Single-cell sequencing provides information that is not confounded by genotypic or phenotypic heterogeneity of bulk samples. Sequencing of one molecular type (RNA, methylated DNA or open chromatin) in a single cell, furthermore, provides insights into the cell’s phenotype and links to its genotype. Nevertheless, only by taking measurements of these phenotypes and genotypes from the same single cells can such inferences be made unambiguously. In this review, we survey the first experimental approaches that assay, in parallel, multiple molecular types from the same single cell, before considering the challenges and opportunities afforded by these and future technologies. Copyright © 2016. Published by Elsevier Ltd.

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September 22, 2019

Genome-wide analysis of complex wheat gliadins, the dominant carriers of celiac disease epitopes.

Gliadins, specified by six compound chromosomal loci (Gli-A1/B1/D1 and Gli-A2/B2/D2) in hexaploid bread wheat, are the dominant carriers of celiac disease (CD) epitopes. Because of their complexity, genome-wide characterization of gliadins is a strong challenge. Here, we approached this challenge by combining transcriptomic, proteomic and bioinformatic investigations. Through third-generation RNA sequencing, full-length transcripts were identified for 52 gliadin genes in the bread wheat cultivar Xiaoyan 81. Of them, 42 were active and predicted to encode 25 a-, 11 ?-, one d- and five ?-gliadins. Comparative proteomic analysis between Xiaoyan 81 and six newly-developed mutants each lacking one Gli locus indicated the accumulation of 38 gliadins in the mature grains. A novel group of a-gliadins (the CSTT group) was recognized to contain very few or no CD epitopes. The d-gliadins identified here or previously did not carry CD epitopes. Finally, the mutant lacking Gli-D2 showed significant reductions in the most celiac-toxic a-gliadins and derivative CD epitopes. The insights and resources generated here should aid further studies on gliadin functions in CD and the breeding of healthier wheat.

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September 22, 2019

Global identification of the full-length transcripts and alternative splicing related to phenolic acid biosynthetic genes in Salvia miltiorrhiza.

Salvianolic acids are among the main bioactive components in Salvia miltiorrhiza, and their biosynthesis has attracted widespread interest. However, previous studies on the biosynthesis of phenolic acids using next-generation sequencing platforms are limited with regard to the assembly of full-length transcripts. Based on hybrid-seq (next-generation and single molecular real-time sequencing) of the S. miltiorrhiza root transcriptome, we experimentally identified 15 full-length transcripts and four alternative splicing events of enzyme-coding genes involved in the biosynthesis of rosmarinic acid. Moreover, we herein demonstrate that lithospermic acid B accumulates in the phloem and xylem of roots, in agreement with the expression patterns of the identified key genes related to rosmarinic acid biosynthesis. According to co-expression patterns, we predicted that six candidate cytochrome P450s and five candidate laccases participate in the salvianolic acid pathway. Our results provide a valuable resource for further investigation into the synthetic biology of phenolic acids in S. miltiorrhiza.

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September 22, 2019

Characterization of fusion genes and the significantly expressed fusion isoforms in breast cancer by hybrid sequencing.

We developed an innovative hybrid sequencing approach, IDP-fusion, to detect fusion genes, determine fusion sites and identify and quantify fusion isoforms. IDP-fusion is the first method to study gene fusion events by integrating Third Generation Sequencing long reads and Second Generation Sequencing short reads. We applied IDP-fusion to PacBio data and Illumina data from the MCF-7 breast cancer cells. Compared with the existing tools, IDP-fusion detects fusion genes at higher precision and a very low false positive rate. The results show that IDP-fusion will be useful for unraveling the complexity of multiple fusion splices and fusion isoforms within tumorigenesis-relevant fusion genes. © The Author(s) 2015. Published by Oxford University Press on behalf of Nucleic Acids Research.

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September 22, 2019

Single-cell RNAseq for the study of isoforms-how is that possible?

Single-cell RNAseq and alternative splicing studies have recently become two of the most prominent applications of RNAseq. However, the combination of both is still challenging, and few research efforts have been dedicated to the intersection between them. Cell-level insight on isoform expression is required to fully understand the biology of alternative splicing, but it is still an open question to what extent isoform expression analysis at the single-cell level is actually feasible. Here, we establish a set of four conditions that are required for a successful single-cell-level isoform study and evaluate how these conditions are met by these technologies in published research.

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September 22, 2019

Long-read sequencing of human cytomegalovirus transcriptome reveals RNA isoforms carrying distinct coding potentials.

The human cytomegalovirus (HCMV) is a ubiquitous, human pathogenic herpesvirus. The complete viral genome is transcriptionally active during infection; however, a large part of its transcriptome has yet to be annotated. In this work, we applied the amplified isoform sequencing technique from Pacific Biosciences to characterize the lytic transcriptome of HCMV strain Towne varS. We developed a pipeline for transcript annotation using long-read sequencing data. We identified 248 transcriptional start sites, 116 transcriptional termination sites and 80 splicing events. Using this information, we have annotated 291 previously undescribed or only partially annotated transcript isoforms, including eight novel antisense transcripts and their isoforms, as well as a novel transcript (RS2) in the short repeat region, partially antisense to RS1. Similarly to other organisms, we discovered a high transcriptional diversity in HCMV, with many transcripts only slightly differing from one another. Comparing our transcriptome profiling results to an earlier ribosome footprint analysis, we have concluded that the majority of the transcripts contain multiple translationally active ORFs, and also that most isoforms contain unique combinations of ORFs. Based on these results, we propose that one important function of this transcriptional diversity may be to provide a regulatory mechanism at the level of translation.

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September 22, 2019

De novo assembly of a Chinese soybean genome.

Soybean was domesticated in China and has become one of the most important oilseed crops. Due to bottlenecks in their introduction and dissemination, soybeans from different geographic areas exhibit extensive genetic diversity. Asia is the largest soybean market; therefore, a high-quality soybean reference genome from this area is critical for soybean research and breeding. Here, we report the de novo assembly and sequence analysis of a Chinese soybean genome for “Zhonghuang 13” by a combination of SMRT, Hi-C and optical mapping data. The assembled genome size is 1.025 Gb with a contig N50 of 3.46 Mb and a scaffold N50 of 51.87 Mb. Comparisons between this genome and the previously reported reference genome (cv. Williams 82) uncovered more than 250,000 structure variations. A total of 52,051 protein coding genes and 36,429 transposable elements were annotated for this genome, and a gene co-expression network including 39,967 genes was also established. This high quality Chinese soybean genome and its sequence analysis will provide valuable information for soybean improvement in the future.

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September 22, 2019

The small peptide world in long noncoding RNAs.

Long noncoding RNAs (lncRNAs) are a group of transcripts that are longer than 200 nucleotides (nt) without coding potential. Over the past decade, tens of thousands of novel lncRNAs have been annotated in animal and plant genomes because of advanced high-throughput RNA sequencing technologies and with the aid of coding transcript classifiers. Further, a considerable number of reports have revealed the existence of stable, functional small peptides (also known as micropeptides), translated from lncRNAs. In this review, we discuss the methods of lncRNA classification, the investigations regarding their coding potential and the functional significance of the peptides they encode.

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September 22, 2019

Quantitative isoform-profiling of highly diversified recognition molecules.

Complex biological systems rely on cell surface cues that govern cellular self-recognition and selective interactions with appropriate partners. Molecular diversification of cell surface recognition molecules through DNA recombination and complex alternative splicing has emerged as an important principle for encoding such interactions. However, the lack of tools to specifically detect and quantify receptor protein isoforms is a major impediment to functional studies. We here developed a workflow for targeted mass spectrometry by selected reaction monitoring (SRM) that permits quantitative assessment of highly diversified protein families. We apply this workflow to dissecting the molecular diversity of the neuronal neurexin receptors and uncover an alternative splicing-dependent recognition code for synaptic ligands.

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