Nanopore long-read RNA sequencing shows region-specific sex differences in wild-type mouse brain mRNA isoform expression and usage
Ontology highlight
ABSTRACT:
ORGANISM(S): Mus musculus
PROVIDER: GSE246705 | GEO | 2024/01/31
REPOSITORIES: GEO
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Project description:Differences in transcript splicing are well documented among tissues and between sexes in humans and other organisms. Further, splicing changes in selected genes have been reported in human and mouse brain development. Using high-throughput RNA sequencing we characterized the transcriptome-wide splicing changes occurring over the lifespan in the human brain. We found that in two brain regions, prefrontal cortex and cerebellum, as many as 37% of all expressed protein-coding multi-exon genes undergo significant splicing changes between birth and 98 years of age. Approximately 40% of these changes take place in aging. We confirmed our findings using exon arrays and PCRs and detected identified splicing changes at the protein level. We further associated distinct patterns of age-related splicing changes with expression of the corresponding splicing factors. Thus, splicing plays an important role in shaping the human brain transcriptome in both development and aging. Human post-mortem brain samples from prefrontal cortex and cerebellum from 30 individuals with ages from birth to 100 years were collected. For each five individuals of similar age RNA, extracted from dissected tissue, was pooled resulting in 5 pooled samples. Obtained RNA was hybridized with Affymetrix Human Exon 1.0 ST arrays.
2013-01-22 | E-GEOD-26586 | biostudies-arrayexpress
Project description:Differences in transcript splicing are well documented among tissues and between sexes in humans and other organisms. Further, splicing changes in selected genes have been reported in human and mouse brain development. Using high-throughput RNA sequencing we characterized the transcriptome-wide splicing changes occurring over the lifespan in the human brain. We found that in two brain regions, prefrontal cortex and cerebellum, as many as 37% of all expressed protein-coding multi-exon genes undergo significant splicing changes between birth and 98 years of age. Approximately 40% of these changes take place in aging. We confirmed our findings using exon arrays and PCRs and detected identified splicing changes at the protein level. We further associated distinct patterns of age-related splicing changes with expression of the corresponding splicing factors. Thus, splicing plays an important role in shaping the human brain transcriptome in both development and aging.
2013-01-22 | GSE26586 | GEO
Project description:In this study we have employed RNA seq on ten different tissues including four brain tissues from two boars to gain a understanding of the differential variations in transcriptional profiles for these tissues consisting of occipital cortex, frontal cortex, hypothalamus and cerebellum along with such diverse tissues as heart, spleen, liver, kidney, lung and musculus longissimus dorsi. This has enabled us to perform comparative gene expression analysis of brain regions versus non-brain tissues along with inter-brain tissue comparisons. Hence, we have tested for differentially expressed genes and isoforms, differential splicing, transcription start sites (TSS), and differential promoter usage between all ten porcine tissues.
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Project description:The transcriptome profiles of the model plant Arabidopsis thaliana have been extensively studied and charcaterised under different developmental and physiological conditions. However, most of these “RNA-sequencing” datasets have been generated using the sequencing of reverse-transcribed cDNAs from mRNAs that have a relatively short read length. Here, we performed direct RNA sequencing using the latest Oxford Nanopore Technology (ONT) with unusual read length. We demonstrate that the complexity of the A. thaliana transcriptomes has been under-estimated. The ONT direct RNA sequencing technology identified transcript isoforms at a vegetative (14 day old seedlings, stage 1.04) and a reproductive stage (stage 6.00-6) when 10% of the flowers had opened. In-house software called TrackCluster was used to determine alternative transcription initiation (ATI), possible alternative polyadenylation (APA), poly(A) length, alternative splicing (AS), and fusion transcripts. Tombo software was used to detect RNA base modifications. More than 38,500 novel transcript isoforms were identified, including six categories of fusion-transcripts which may result from differential RNA processing mechanisms. Fusion-transcripts are prone to mis-assembly by sequencing with short reads using next-generation-sequencing (NGS). These new transcript isoforms provide important additions to the annotated Arabidopsis genome. The power of ONT in detecting RNA modifications was demonstrated by characterisation of the modifications between mobile mRNAs and total mRNAs. The mobile mRNAs were enriched in m5C modifications, which is consistent with a recent finding that m5C modification in mRNAs is crucial for their long-distance movement. In summary, ONT direct RNA sequencing greatly enhances the identification of novel RNA transcript isoforms and RNA base modifications.
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Project description:Alternative splicing contributes to transcriptomic complexity and plays a role in the regulation of cellular identity and function, but the correct assembly of transcripts of complex loci as well as their quantification based on short-read sequencing is non-trivial. Recent long-read sequencing methods such as those from ONT and PacBio overcome these problems by potentially sequencing full transcripts. The activation of brown adipose tissue e.g., by reduced ambient temperature (cold) exposure, positively affects metabolism by increasing energy expenditure and releasing endocrine factors and has been shown to involve specific alternative splicing events. Here we assessed important features of ONT long read sequencing protocols in relation to Illumina short read sequencing: (i) Alignment characteristics to the reference genome and transcriptome, (ii) Gene and transcript detection and quantification, (iii) Detection of differential gene and transcript expression events, (iv) Transcriptome reannotation and (v) Detection of differential transcript usage events. We find that ONT long-read sequencing is advantageous in terms of transcriptome reassembly, especially when the reads are enriched for full length reads. Illumina sequencing, due to the higher number of counts available, has a higher statistical power for calling differentiall expressed/used features, whereas long-read sequencing has a lower risk of calling false positive events due to the better ability to unambiguously map reads to transcripts. Finally we describe novel transcript isoforms in cold-activated murine iBAT reassembled from ONT long reads.
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Project description:Alternative splicing contributes to transcriptomic complexity and plays a role in the regulation of cellular identity and function, but the correct assembly of transcripts of complex loci as well as their quantification based on short-read sequencing is non-trivial. Recent long-read sequencing methods such as those from ONT and PacBio overcome these problems by potentially sequencing full transcripts. The activation of brown adipose tissue e.g., by reduced ambient temperature (cold) exposure, positively affects metabolism by increasing energy expenditure and releasing endocrine factors and has been shown to involve specific alternative splicing events. Here we assessed important features of ONT long read sequencing protocols in relation to Illumina short read sequencing: (i) Alignment characteristics to the reference genome and transcriptome, (ii) Gene and transcript detection and quantification, (iii) Detection of differential gene and transcript expression events, (iv) Transcriptome reannotation and (v) Detection of differential transcript usage events. We find that ONT long-read sequencing is advantageous in terms of transcriptome reassembly, especially when the reads are enriched for full length reads. Illumina sequencing, due to the higher number of counts available, has a higher statistical power for calling differentiall expressed/used features, whereas long-read sequencing has a lower risk of calling false positive events due to the better ability to unambiguously map reads to transcripts. Finally we describe novel transcript isoforms in cold-activated murine iBAT reassembled from ONT long reads.
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