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Nanopore sequencing unveils the complexity of the murine brown adipose tissue transcriptome.

ABSTRACT: This SuperSeries is composed of the SubSeries listed below.

ORGANISM(S): Mus musculus

PROVIDER: GSE212574 | GEO | 2023/09/18

REPOSITORIES: GEO

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Nanopore sequencing unveils the complexity of the cold-activated murine brown adipose tissue transcriptome.

Project description:Alternative transcription increases transcriptome complexity by expression of multiple transcripts per gene. Annotation and quantification of transcripts using short-read sequencing is non-trivial. Long-read sequencing aims at overcoming these problems by sequencing full-length transcripts. Activation of brown adipose tissue (BAT) thermogenesis involves major transcriptomic remodeling and positively affects metabolism via increased energy expenditure. We benchmark Oxford Nanopore Technology (ONT) long-read sequencing protocols to Illumina short-read sequencing assessing alignment characteristics, gene and transcript detection and quantification, differential gene and transcript expression, transcriptome reannotation, and differential transcript usage (DTU). We find ONT sequencing is superior to Illumina for transcriptome reassembly, reducing the risk of false-positive events by unambiguously mapping reads to transcripts. We identified novel isoforms of genes undergoing DTU in cold-activated BAT including Cars2, Adtrp, Acsl5, Scp2, Aldoa, and Pde4d, validated by real-time PCR. The reannotated murine BAT transcriptome established here provides a framework for future investigations into the regulation of BAT.

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Nanopore sequencing unveils the complexity of the murine brown adipose tissue transcriptome.

Project description:Nanopore sequencing unveils the complexity of the murine brown adipose tissue transcriptome.

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Nanopore sequencing unveils the complexity of the murine brown adipose tissue transcriptome: Illumina RNA-Seq of iBAT from cold treated mice [01_iBAT_illumina]

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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Nanopore sequencing unveils the complexity of the murine brown adipose tissue transcriptome: Illumina RNA-Seq of eWAT from cold treated mice [05_eWAT_illumina]

2023-09-18 | GSE212573 | GEO

Nanopore sequencing unveils the complexity of the murine brown adipose tissue transcriptome: ONT direct RNA-Seq of iBAT from cold treated mice [02_iBAT_rna]

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Nanopore sequencing unveils the complexity of the murine brown adipose tissue transcriptome: ONT direct cDNA-Seq of iBAT from cold treated mice [04_iBAT_cdna]

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Nanopore sequencing unveils the complexity of the murine brown adipose tissue transcriptome: ONT PCR RNA-Seq (TeloPrime) of iBAT from cold treated mice [03_iBAT_teloprime]

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Nanopore sequencing unveils the complexity of the murine brown adipose tissue transcriptome: Illumina RNA-Seq of eWAT from cold treated mice [05_eWAT_illumina]

Project description:Nanopore sequencing unveils the complexity of the murine brown adipose tissue transcriptome: Illumina RNA-Seq of eWAT from cold treated mice [05_eWAT_illumina]

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Nanopore sequencing unveils the complexity of the murine brown adipose tissue transcriptome: ONT direct RNA-Seq of iBAT from cold treated mice [02_iBAT_rna]

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