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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.


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.


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.


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.


September 22, 2019  |  

Event analysis: Using transcript events to improve estimates of abundance in RNA-seq data.

Alternative splicing leverages genomic content by allowing the synthesis of multiple transcripts and, by implication, protein isoforms, from a single gene. However, estimating the abundance of transcripts produced in a given tissue from short sequencing reads is difficult and can result in both the construction of transcripts that do not exist, and the failure to identify true transcripts. An alternative approach is to catalog the events that make up isoforms (splice junctions and exons). We present here the Event Analysis (EA) approach, where we project transcripts onto the genome and identify overlapping/unique regions and junctions. In addition, all possible logical junctions are assembled into a catalog. Transcripts are filtered before quantitation based on simple measures: the proportion of the events detected, and the coverage. We find that mapping to a junction catalog is more efficient at detecting novel junctions than mapping in a splice aware manner. We identify 99.8% of true transcripts while iReckon identifies 82% of the true transcripts and creates more transcripts not included in the simulation than were initially used in the simulation. Using PacBio Iso-seq data from a mouse neural progenitor cell model, EA detects 60% of the novel junctions that are combinations of existing exons while only 43% are detected by STAR. EA further detects ~5,000 annotated junctions missed by STAR. Filtering transcripts based on the proportion of the transcript detected and the number of reads on average supporting that transcript captures 95% of the PacBio transcriptome. Filtering the reference transcriptome before quantitation, results in is a more stable estimate of isoform abundance, with improved correlation between replicates. This was particularly evident when EA is applied to an RNA-seq study of type 1 diabetes (T1D), where the coefficient of variation among subjects (n = 81) in the transcript abundance estimates was substantially reduced compared to the estimation using the full reference. EA focuses on individual transcriptional events. These events can be quantitate and analyzed directly or used to identify the probable set of expressed transcripts. Simple rules based on detected events and coverage used in filtering result in a dramatic improvement in isoform estimation without the use of ancillary data (e.g., ChIP, long reads) that may not be available for many studies. Copyright © 2018 Newman et al.


September 22, 2019  |  

Transcription-associated mutation promotes RNA complexity in highly expressed genes – A major new source of selectable variation.

Alternatively spliced transcript isoforms are thought to play a critical role for functional diversity. However, the mechanism generating the enormous diversity of spliced transcript isoforms remains unknown, and its biological significance remains unclear. We analyzed transcriptomes in saker falcons, chickens, and mice to show that alternative splicing occurs more frequently, yielding more isoforms, in highly expressed genes. We focused on hemoglobin in the falcon, the most abundantly expressed genes in blood, finding that alternative splicing produces 10-fold more isoforms than expected from the number of splice junctions in the genome. These isoforms were produced mainly by alternative use of de novo splice sites generated by transcription-associated mutation (TAM), not by the RNA editing mechanism normally invoked. We found that high expression of globin genes increases mutation frequencies during transcription, especially on nontranscribed DNA strands. After DNA replication, transcribed strands inherit these somatic mutations, creating de novo splice sites, and generating multiple distinct isoforms in the cell clone. Bisulfate sequencing revealed that DNA methylation may counteract this process by suppressing TAM, suggesting DNA methylation can spatially regulate RNA complexity. RNA profiling showed that falcons living on the high Qinghai-Tibetan Plateau possess greater global gene expression levels and higher diversity of mean to high abundance isoforms (reads per kilobases per million mapped reads?=18) than their low-altitude counterparts, and we speculate that this may enhance their oxygen transport capacity under low-oxygen environments. Thus, TAM-induced RNA diversity may be physiologically significant, providing an alternative strategy in lifestyle evolution.


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