Partitioning RNAs by length improves transcriptome reconstruction from short-read RNA-seq data

Ringeling, Francisca Rojas; Chakraborty, Shounak; Vissers, Caroline; Reiman, Derek; Patel, Akshay; Lee, Ki-Heon; Hong, Ari; Park, Chanwoo; Reska, Tim; Gagneur, Julien; Chang, Hyeshik; Spletter, Maria L.; Yoon, Ki‐Jun; Ming, Guo Li; Song, Hongjun; Canzar, Stefan

doi:10.1038/s41587-021-01136-7

Cited by 10 publications

(8 citation statements)

References 68 publications

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“…7A). We found little overlap between stringtie and FLAIR reannoations in line with other reports (Ringeling et al, 2022). Among the genes with significant isoform switches between cold-activated BAT compared to the controls, we observed phosphodiesterase 4D ( Pde4 d), regulating levels of the signalling intermediate cAMP, which activates lipolysis, glucose uptake, and thermogenesis in brown adipocytes (Reverte-Salisa et al, 2019); the thermogenesis regulating hydrolase androgen dependent TFPI regulating protein ( Adtrp; P. Li et al, 2022) and regulators of fatty acid metabolism (acyl-CoA synthetase long-chain family member 5; Acsl5 ), glycolysis (Aldolase A; Aldoa ), protein sorting (endoplasmic reticulum-golgi intermediate compartment 1; Ergic 1), lipid synthesis (MLX interacting protein-like; Mlxip l), beta-oxidation (sterol carrier protein 2, liver; Scp2 ) and protein cysteinylation (cysteinyl-tRNA synthetase gene; Cars2; Akaike et al, 2017; Fig.…”

Section: Resultssupporting

confidence: 92%

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

Engelhard

Khani

Derdak

et al. 2022

Preprint

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Alternative transcription increases transcriptome complexity by expression of multiple transcripts per gene and thus fine tunes cellular identity and function. Annotation and quantification of transcripts at complex loci using short-read sequencing is non-trivial. Recent long-read sequencing methods such as those from Oxford Nanopore Technologies (ONT) and Pacific Biosciences aim at overcoming these problems by sequencing full length transcripts. Activation of BAT thermogenesis involves major transcriptomic remodelling and positively affects metabolism via increased energy expenditure and endocrine factors. Here we comprehensively benchmark features of ONT long-read sequencing protocols compared to Illumina shortread sequencing assessing alignment characteristics, gene and transcript detection and quantification, differential gene and transcript expression, transcriptome reannotation and differential transcript usage (DTU). We find that ONT sequencing is superior to Illumina for transcriptome reassembly and reduces the risk of false-positive events due to the ability to unambiguously map reads to transcripts, at the expense of statistical power for calling differentially expressed features. We identified novel isoforms of genes undergoing DTU in cold-activated BAT including Cars2, Adtrp, Acsl5, Scp2, Aldoa and Pde4d, validated by RT-qPCR. Finally, we provide a reannotation of the murine iBAT transcriptome as a valuable resource for researchers interested in the molecular biology underlying the regulation of BAT.

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Section: Resultssupporting

confidence: 92%

Section: Differential Transcript Usage Analysis Unravels Gene Express...supporting

confidence: 91%

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

Engelhard

Khani

Derdak

et al. 2022

Preprint

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“…Detailed analysis of individual isoforms has been limited to highly studied genes because studying individual RNA isoforms via high-throughput methods has been challenging since short-read data relies on heuristics to assemble isoforms [7][8][9] , but heuristics cannot outperform directly measuring the trait in question. In principle, long reads have the potential to sequence entire isoforms, providing a direct measurement for each isoform, but long-read data is still far from perfect 10 and also requires its own heuristics-specifically to estimate expression for each isoform 10,11 .…”

Section: Mainmentioning

confidence: 99%

Using deep long-read RNAseq in Alzheimer’s disease brain to assess medical relevance of RNA isoform diversity

Heberle,

Brandon,

Page

et al. 2023

Preprint

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Due to alternative splicing, human protein-coding genes average over eight RNA isoforms, resulting in nearly four distinct protein coding sequences per gene. Long-read RNAseq (IsoSeq) enables more accurate quantification of isoforms, shedding light on their specific roles. To assess the clinical relevance of measuring RNA isoform expression, we sequenced 12 aged human frontal cortices (6 Alzheimer's disease cases and 6 controls, 50% female) using one Oxford Nanopore PromethION flow cell per sample. Our study uncovered 53 new high-confidence RNA isoforms in clinically relevant genes, including several where the new isoform was one of the most highly expressed for that gene. Specific examples include WDR4 (61%; microcephaly), MYL3 (44%; hypertrophic cardiomyopathy), and MTHFS (25%; major depression, schizophrenia, bipolar disorder). Other notable genes with new high-confidence isoforms include CPLX2 (10%; schizophrenia, epilepsy) and MAOB (9%; targeted for Parkinson's disease treatment). We identified 1,917 clinically relevant genes expressing multiple isoforms in human frontal cortex, where 1,018 had multiple isoforms with different protein coding sequences, demonstrating the need to better understand how individual isoforms from a "single" gene are involved in human health and disease. Exactly 98 of the 1,917 genes are implicated in brain-related diseases, including Alzheimer's disease genes such as APP (A-beta; precursor protein; five), MAPT (tau protein; four), and BIN1 (eight). We also found 99 differentially expressed RNA isoforms between Alzheimer's cases and controls, despite the genes themselves not exhibiting differential expression. Our findings highlight the significant knowledge gaps in RNA isoform diversity and their clinical relevance. Deep long-read RNA sequencing will be necessary going forward to fully comprehend the clinical relevance of individual isoforms for a "single" gene. Key words: RNA isoforms, Clinical relevance, Long reads, Nanopore sequencing, Alzheimer's disease, Human brain.

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“…Ongoing attempts to develop laboratory methods, segregating transcripts by length prior to processing and sequencing, partly serves to illustrate the wide acceptance that existing methods to model transcript-level data need improved. [242] Both the HTA 2.0 and the updated version, the Clariom D array, contain probes designed to span individual exons ("junction probes") in known transcripts, and these were intended to improve the detection of AS of cassette exons. [98] There are very few methods for studying AS/AEU using high-density arrays, [243] with only one making direct use of these exon-exon junction spanning probes, [244] and one which has subsequently been adapted for RNA-seq.…”

Section: Modeling Alternative Splicing With Bulk Transcriptomicsmentioning

confidence: 99%

Section: Modeling Alternative Splicing With Bulk Transcriptomicsmentioning

confidence: 99%

Transcriptomics for Clinical and Experimental Biology Research: Hang on a Seq

et al. 2023

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Sequencing the human genome empowers translational medicine, facilitating transcriptome-wide molecular diagnosis, pathway biology, and drug repositioning. Initially, microarrays are used to study the bulk transcriptome; but now short-read RNA sequencing (RNA-seq) predominates. Positioned as a superior technology, that makes the discovery of novel transcripts routine, most RNA-seq analyses are in fact modeled on the known transcriptome. Limitations of the RNA-seq methodology have emerged, while the design of, and the analysis strategies applied to, arrays have matured. An equitable comparison between these technologies is provided, highlighting advantages that modern arrays hold over RNA-seq. Array protocols more accurately quantify constitutively expressed protein coding genes across tissue replicates, and are more reliable for studying lower expressed genes. Arrays reveal long noncoding RNAs (lncRNA) are neither sparsely nor lower expressed than protein coding genes. Heterogeneous coverage of constitutively expressed genes observed with RNA-seq, undermines the validity and reproducibility of pathway analyses. The factors driving these observations, many of which are relevant to long-read or single-cell sequencing are discussed. As proposed herein, a reappreciation of bulk transcriptomic methods is required, including wider use of the modern high-density array data-to urgently revise existing anatomical RNA reference atlases and assist with more accurate study of lncRNAs.

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Partitioning RNAs by length improves transcriptome reconstruction from short-read RNA-seq data

Cited by 10 publications

References 68 publications

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

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

Using deep long-read RNAseq in Alzheimer’s disease brain to assess medical relevance of RNA isoform diversity

Transcriptomics for Clinical and Experimental Biology Research: Hang on a Seq

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