Full-Length Transcriptome Sequencing Reveals Alternative Splicing and lncRNA Regulation during Nodule Development in Glycine max.
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ABSTRACT:
SUBMITTER: Liu J
PROVIDER: S-EPMC9266934 | biostudies-literature |
REPOSITORIES: biostudies-literature
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Project description:IntroductionPeanut (Arachis hypogaea L.), also called groundnut is an important oil and cash crop grown widely in the world. The annual global production of groundnuts has increased to approximately 50 million tons, which provides a rich source of vegetable oils and proteins for humans. Low temperature (non-freezing) is one of the major factors restricting peanut growth, yield, and geographic distribution. Since the complexity of cold-resistance trait, the molecular mechanism of cold tolerance and related gene networks were largely unknown in peanut.MethodsIn this study, comparative transcriptomic analysis of two peanut cultivars (SLH vs. ZH12) with differential cold tolerance under low temperature (10°C) was performed using Oxford Nanopore Technology (ONT) platform.Results and discussionAs a result, we identified 8,949 novel gene loci and 95,291 new/novel isoforms compared with the reference database. More differentially expressed genes (DEGs) were discovered in cold-sensitive cultivar (ZH12) than cold-tolerant cultivar (SLH), while more alternative splicing events were found in SLH compared to ZH12. Gene Ontology (GO) analyses of the common DEGs showed that the "response to stress", "chloroplast part", and "transcription factor activity" were the most enriched GO terms, indicating that photosynthesis process and transcription factors play crucial roles in cold stress response in peanut. We also detected a total of 708 differential alternative splicing genes (DASGs) under cold stress compared to normal condition. Intron retention (IR) and exon skipping (ES) were the most prevalent alternative splicing (AS) events. In total, 4,993 transcription factors and 292 splicing factors were detected, many of them had differential expression levels and/or underwent AS events in response to cold stress. Overexpression of two candidate genes (encoding trehalose-6-phosphatephosphatases, AhTPPs) in yeast improves cold tolerance. This study not only provides valuable resources for the study of cold resistance in peanut but also lay a foundation for genetic modification of cold regulators to enhance stress tolerance in crops.
| S-EPMC10954824 | biostudies-literature
Project description:Quinoa is a cold-resistant and nutrient-rich crop. To decipher the cold stress response of quinoa, the full-length transcriptomes of the cold-resistant quinoa variety CRQ64 and the cold-sensitive quinoa variety CSQ5 were compared. We identified 55,389 novel isoforms and 6432 novel genes in these transcriptomes. Under cold stress, CRQ64 had more differentially expressed genes (DEGs) and differentially alternative splicing events compared to non-stress conditions than CSQ5. DEGs that were specifically present only in CRQ64 were significantly enriched in processes which contribute to osmoregulation and ROS homeostasis in plants, such as sucrose metabolism and phenylpropanoid biosynthesis. More genes with differential alternative splicing under cold stress were enriched in peroxidase functions in CRQ64. In total, 5988 transcription factors and 2956 long non-coding RNAs (LncRNAs) were detected in this dataset. Many of these had altered expression patterns under cold stress compared to non-stress conditions. Our transcriptome results demonstrate that CRQ64 undergoes a wider stress response than CSQ5 under cold stress. Our results improved the annotation of the quinoa genome and provide new insight into the mechanisms of cold resistance in quinoa.
| S-EPMC9144462 | biostudies-literature
Project description:BackgroundThe vast diversification of proteins in eukaryotic cells has been related with multiple transcript isoforms from a single gene that result in alternative splicing (AS) of primary transcripts. Analysis of RNA sequencing data from expressed sequence tags and next generation RNA sequencing has been crucial for AS identification and genome-wide AS studies. For the identification of AS events from the related legume species Phaseolus vulgaris and Glycine max, 157 and 88 publicly available RNA-seq libraries, respectively, were analyzed.ResultsWe identified 85,570 AS events from P. vulgaris in 72% of expressed genes and 134,316 AS events in 70% of expressed genes from G. max. These were categorized in seven AS event types with intron retention being the most abundant followed by alternative acceptor and alternative donor, representing ~75% of all AS events in both plants. Conservation of AS events in homologous genes between the two species was analyzed where an overrepresentation of AS affecting 5'UTR regions was observed for certain types of AS events. The conservation of AS events was experimentally validated for 8 selected genes, through RT-PCR analysis. The different types of AS events also varied by relative position in the genes. The results were consistent in both species.ConclusionsThe identification and analysis of AS events are first steps to understand their biological relevance. The results presented here from two related legume species reveal high conservation, over ~15-20 MY of divergence, and may point to the biological relevance of AS.
| S-EPMC5568362 | biostudies-literature
Project description:Alternative splicing is a post-transcriptional regulatory mechanism producing distinct mRNA molecules from a single pre-mRNA with a prominent role in the development and function of the central nervous system. We used long-read isoform sequencing to generate full-length transcript sequences in the human and mouse cortex. We identify novel transcripts not present in existing genome annotations, including transcripts mapping to putative novel (unannotated) genes and fusion transcripts incorporating exons from multiple genes. Global patterns of transcript diversity are similar between human and mouse cortex, although certain genes are characterized by striking differences between species. We also identify developmental changes in alternative splicing, with differential transcript usage between human fetal and adult cortex. Our data confirm the importance of alternative splicing in the cortex, dramatically increasing transcriptional diversity and representing an important mechanism underpinning gene regulation in the brain. We provide transcript-level data for human and mouse cortex as a resource to the scientific community.
| S-EPMC8609283 | biostudies-literature
Project description:Long-read sequencing holds great potential for transcriptome analysis because it offers researchers an affordable method to annotate the transcriptomes of non-model organisms. This, in turn, will greatly benefit future work on less-researched organisms like unicellular eukaryotes that cannot rely on large consortia to generate these transcriptome annotations. However, to realize this potential, several remaining molecular and computational challenges will have to be overcome. In this review, we have outlined the limitations of short-read sequencing technology and how long-read sequencing technology overcomes these limitations. We have also highlighted the unique challenges still present for long-read sequencing technology and provided some suggestions on how to overcome these challenges going forward. This article is part of a discussion meeting issue 'Single cell ecology'.
| S-EPMC6792442 | biostudies-literature
Project description:BackgroundGossypium australe F. Mueller (2n = 2x = 26, G2 genome) possesses valuable characteristics. For example, the delayed gland morphogenesis trait causes cottonseed protein and oil to be edible while retaining resistance to biotic stress. However, the lack of gene sequences and their alternative splicing (AS) in G. australe remain unclear, hindering to explore species-specific biological morphogenesis.ResultsHere, we report the first sequencing of the full-length transcriptome of the Australian wild cotton species, G. australe, using Pacific Biosciences single-molecule long-read isoform sequencing (Iso-Seq) from the pooled cDNA of ten tissues to identify transcript loci and splice isoforms. We reconstructed the G. australe full-length transcriptome and identified 25,246 genes, 86 pre-miRNAs and 1468 lncRNAs. Most genes (12,832, 50.83%) exhibited two or more isoforms, suggesting a high degree of transcriptome complexity in G. australe. A total of 31,448 AS events in five major types were found among the 9944 gene loci. Among these five major types, intron retention was the most frequent, accounting for 68.85% of AS events. 29,718 polyadenylation sites were detected from 14,536 genes, 7900 of which have alternative polyadenylation sites (APA). In addition, based on our AS events annotations, RNA-Seq short reads from germinating seeds showed that differential expression of these events occurred during seed germination. Ten AS events that were randomly selected were further confirmed by RT-PCR amplification in leaf and germinating seeds.ConclusionsThe reconstructed gene sequences and their AS in G. australe would provide information for exploring beneficial characteristics in G. australe.
| S-EPMC6701088 | biostudies-literature
Project description:Huntington's disease (HD) is an autosomal dominant neurodegenerative disorder caused by a CAG expansion in the gene-encoding Huntingtin (HTT). Transcriptome dysregulation is a major feature of HD pathogenesis, as revealed by a large body of work on gene expression profiling of tissues from human HD patients and mouse models. These studies were primarily focused on transcriptional changes affecting steady-state overall gene expression levels using microarray based approaches. A major missing component, however, has been the study of transcriptome changes at the post-transcriptional level, such as alternative splicing. Alternative splicing is a critical mechanism for expanding regulatory and functional diversity from a limited number of genes, and is particularly complex in the mammalian brain. Here we carried out a deep RNA-seq analysis of the BA4 (Brodmann area 4) motor cortex from seven human HD brains and seven controls to systematically discover aberrant alternative splicing events and characterize potential associated splicing factors in HD. We identified 593 differential alternative splicing events between HD and control brains. Using two expanded panels with a total of 108 BA4 tissues from patients and controls, we identified four splicing factors exhibiting significantly altered expression levels in HD patient brains. Moreover, follow-up molecular analyses of one splicing factor PTBP1 revealed its impact on disease-associated splicing patterns in HD. Collectively, our data provide genomic evidence for widespread splicing dysregulation in HD brains, and suggest the role of aberrant alternative splicing in the pathogenesis of HD.
| S-EPMC5179942 | biostudies-literature
Project description:Seagrass meadows are among the four most productive marine ecosystems in the world. Zostera japonica (Z. japonica) is the most widely distributed species of seagrass in China. However, there is no reference genome or transcriptome available for Z. japonica, impeding progress in functional genomic and molecular ecology studies in this species. Temperature is the main factor that controls the distribution and growth of seagrass around the world, yet how seagrass responds to heat stress remains poorly understood due to the lack of genomic and transcriptomic data. In this study, we applied a combination of second- and third-generation sequencing technologies to sequence full-length transcriptomes of Z. japonica. In total, we obtained 58,134 uniform transcripts, which included 46,070 high-quality full-length transcript sequences. We identified 15,411 simple sequence repeats, 258 long non-coding RNAs and 28,038 open reading frames. Exposure to heat elicited a complex transcriptional response in genes involved in posttranslational modification, protein turnover and chaperones. Overall, our study provides the first large-scale full-length trascriptome in Zostera japonica, allowing for structural, functional and comparative genomics studies in this important seagrass species. Although previous studies have focused specifically on heat shock proteins, we found that examination of other heat stress related genes is important for studying response to heat stress in seagrass. This study provides a genetic resource for the discovery of genes related to heat stress tolerance in this species. Our transcriptome can be further utilized in future studies to understand the molecular adaptation to heat stress in Zostera japonica.
| S-EPMC6787188 | biostudies-literature
Project description:Tokay Gecko (Gekko gecko) is a rare and endangered medicinal animal in China. Its dry body has been used as an anti-asthmatic agent for two thousand years. To date, the genome and transcriptome of this species remain poorly understood. Here, we adopted single molecule real-time (SMRT) sequencing to obtain full-length transcriptome data and characterized the transcriptome structure. We identified 882,273 circular consensus (CCS) reads, including 746,317 full-length nonchimeric (FLNC) reads. The transcript cluster analysis revealed 212,964 consensus sequences, including 203,994 high-quality isoforms. In total, 111,372 of 117,888 transcripts were successfully annotated against eight databases (Nr, eggNOG, Swiss-Prot, GO, COG, KOG, Pfam and KEGG). Furthermore, 23,877 alternative splicing events, 169,128 simple sequence repeats (SSRs), 10,437 lncRNAs and 7,932 transcription factors were predicted across all transcripts. To our knowledge, this report is the first to document the G. gecko transcriptome using SMRT sequencing. The full-length transcript data might accelerate transcriptome research and lay the foundation for further research on G. gecko.
| S-EPMC8880673 | biostudies-literature
Project description:BackgroundCancers exhibit complex transcriptomes with aberrant splicing that induces isoform-level differential expression compared to non-diseased tissues. Transcriptomic profiling using short-read sequencing has utility in providing a cost-effective approach for evaluating isoform expression, although short-read assembly displays limitations in the accurate inference of full-length transcripts. Long-read RNA sequencing (Iso-Seq), using the Pacific Biosciences (PacBio) platform, can overcome such limitations by providing full-length isoform sequence resolution which requires no read assembly and represents native expressed transcripts. A constraint of the Iso-Seq protocol is due to fewer reads output per instrument run, which, as an example, can consequently affect the detection of lowly expressed transcripts. To address these deficiencies, we developed a concatenation workflow, PacBio Full-Length Isoform Concatemer Sequencing (PB_FLIC-Seq), designed to increase the number of unique, sequenced PacBio long-reads thereby improving overall detection of unique isoforms. In addition, we anticipate that the increase in read depth will help improve the detection of moderate to low-level expressed isoforms.ResultsIn sequencing a commercial reference (Spike-In RNA Variants; SIRV) with known isoform complexity we demonstrated a 3.4-fold increase in read output per run and improved SIRV recall when using the PB_FLIC-Seq method compared to the same samples processed with the Iso-Seq protocol. We applied this protocol to a translational cancer case, also demonstrating the utility of the PB_FLIC-Seq method for identifying differential full-length isoform expression in a pediatric diffuse midline glioma compared to its adjacent non-malignant tissue. Our data analysis revealed increased expression of extracellular matrix (ECM) genes within the tumor sample, including an isoform of the Secreted Protein Acidic and Cysteine Rich (SPARC) gene that was expressed 11,676-fold higher than in the adjacent non-malignant tissue. Finally, by using the PB_FLIC-Seq method, we detected several cancer-specific novel isoforms.ConclusionThis work describes a concatenation-based methodology for increasing the number of sequenced full-length isoform reads on the PacBio platform, yielding improved discovery of expressed isoforms. We applied this workflow to profile the transcriptome of a pediatric diffuse midline glioma and adjacent non-malignant tissue. Our findings of cancer-specific novel isoform expression further highlight the importance of long-read sequencing for characterization of complex tumor transcriptomes.
| S-EPMC10823626 | biostudies-literature