Improving the diversity of captured full-length isoforms using a normalized single-molecule RNA-sequencing method

Hu, Yueming; Shu, Xing-sheng; Yu, Jiaxian; Sun, Ming-an; Chen, Zewei; Liu, Xianming; Fang, Qiongfang; Zhang, Wei; Hui, Xinjie; Ying, Ying; Fu, Li; Lu, Desheng; Kumar, Rakesh; Wang, Yejun

doi:10.1038/s42003-020-01125-7

Cited by 9 publications

(11 citation statements)

References 48 publications

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“…Despite a clear benefit in expanding the repertoire of gene models, “quantitative” information, such as changes of expression levels, would be lost by cDNA normalization. Differential gene expression results indeed can be more readily possible with data generated from non-normalized Iso-seq libraries but discordant gene expression changes between short- and long-read sequencing platforms have been reported previously [ 68 ]. Indeed, as compared with RNA-seq, the non-normalized Iso-seq dataset prepared from tissues of the same developmental stages gave a skewed representation of abundant transcripts (Addition file 1 : Fig.…”

Section: Discussionmentioning

confidence: 83%

An improved repertoire of splicing variants and their potential roles in Arabidopsis photomorphogenic development

Huang

Lin

2022

Genome Biol

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Background Light switches on the photomorphogenic development of young plant seedlings, allowing young seedlings to acquire photosynthetic capacities and gain survival fitness. Light regulates gene expression at all levels of the central dogma, including alternative splicing (AS) during the photomorphogenic development. However, accurate determination of full-length (FL) splicing variants has been greatly hampered by short-read RNA sequencing technologies. Result In this study, we adopt PacBio isoform sequencing (Iso-seq) to overcome the limitation of the short-read RNA-seq technologies. Normalized cDNA libraries used for Iso-seq allows for comprehensive and effective identification of FL AS variants. Our analyses reveal more than 30,000 splicing variant models from approximately 16,500 gene loci and additionally identify approximately 700 previously unannotated genes. Among the variants, approximately 12,000 represent new gene models. Intron retention (IR) is the most frequently observed form of variants, and many IR-containing AS variants show evidence of engagement in translation. Our study reveals the formation of heterodimers of transcription factors composed of annotated and IR-containing AS variants. Moreover, transgenic plants overexpressing the IR forms of two B-BOX DOMAIN PROTEINs exhibits light-hypersensitive phenotypes, suggesting their regulatory roles in modulating optimal light responses. Conclusions This study provides an accurate and comprehensive portrait of full-length transcript isoforms and experimentally confirms the presence of de novo synthesized AS variants that impose regulatory functions in photomorphogenic development in Arabidopsis.

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Section: Discussionmentioning

confidence: 83%

An improved repertoire of splicing variants and their potential roles in Arabidopsis photomorphogenic development

Huang

Lin

2022

Genome Biol

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“…For each type of tissue, more refined algorithms are needed to further ensure that the SEGs from diseased and normal samples are with the same level of expression. While the current study only observed the stable expression of genes at the single-cell level, recent new techniques have been developed that could identify and quantify the expression of single-cell isoforms and even allele-specific isoforms [ 25 , 37 , 38 , 39 ]. Therefore, it would also be interesting to further the single-cell stable expression analysis on transcripts and allele-specific isoforms.…”

Section: Discussionmentioning

confidence: 99%

“…( C ) Expression of a representative spatial-temporal SEG, EIF1 , in single-cell clusters of representative samples. One sample was selected randomly for each of the adult lung, adult stomach, fetal lung and fetal stomach tissues, respectively [ 25 ]. The single cells were clustered according to the gene expression profile, and shown as points in a two-dimension plane of Uniform Manifold Approximation and Projection (UMAP) [ 26 ].…”

Section: Figurementioning

confidence: 99%

Identification of Human Global, Tissue and Within-Tissue Cell-Specific Stably Expressed Genes at Single-Cell Resolution

Qiu

Chen

Zheng

et al. 2022

IJMS

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Stably Expressed Genes (SEGs) are a set of genes with invariant expression. Identification of SEGs, especially among both healthy and diseased tissues, is of clinical relevance to enable more accurate data integration, gene expression comparison and biomarker detection. However, it remains unclear how many global SEGs there are, whether there are development-, tissue- or cell-specific SEGs, and whether diseases can influence their expression. In this research, we systematically investigate human SEGs at single-cell level and observe their development-, tissue- and cell-specificity, and expression stability under various diseased states. A hierarchical strategy is proposed to identify a list of 408 spatial-temporal SEGs. Development-specific SEGs are also identified, with adult tissue-specific SEGs enriched with the function of immune processes and fetal tissue-specific SEGs enriched in RNA splicing activities. Cells of the same type within different tissues tend to show similar SEG composition profiles. Diseases or stresses do not show influence on the expression stableness of SEGs in various tissues. In addition to serving as markers and internal references for data normalization and integration, we examine another possible application of SEGs, i.e., being applied for cell decomposition. The deconvolution model could accurately predict the fractions of major immune cells in multiple independent testing datasets of peripheral blood samples. The study provides a reliable list of human SEGs at the single-cell level, facilitates the understanding on the property of SEGs, and extends their possible applications.

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“…Due to the identifiability of isoform expression inference ( Hiller et al, 2009 ) and large uncertainty introduced in the process ( Jiang and Wong, 2009 ), isoform-level analysis did not show clear advantages over gene-level analysis in expression changes between the two stages. With the rapid development and wide acceptance of the third-generation long-read sequencing technologies (including PacBio and Nanopore), full-length transcriptome sequencing ( Wang et al, 2019 ; Hu et al, 2020 ) would offer more direct information for isoform-level analysis. Most recently, methods have been developed to sequence poly(A)-tail inclusive full-length transcriptome using the PacBio platform ( Legnini et al, 2019 ; Liu et al, 2019 ), which would be extremely helpful for studying the detailed molecular mechanisms during the dynamic process of oocyte maturation.…”

Section: Discussionmentioning

confidence: 99%

Pervasive 3′-UTR Isoform Switches During Mouse Oocyte Maturation

Chen

Zhang

et al. 2021

Front. Mol. Biosci.

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Oocyte maturation is the foundation for developing healthy individuals of mammals. Upon germinal vesicle breakdown, oocyte meiosis resumes and the synthesis of new transcripts ceases. To quantitatively profile the transcriptomic dynamics after meiotic resumption throughout the oocyte maturation, we generated transcriptome sequencing data with individual mouse oocytes at three main developmental stages: germinal vesicle (GV), metaphase I (MI), and metaphase II (MII). When clustering the sequenced oocytes, results showed that isoform-level expression analysis outperformed gene-level analysis, indicating isoform expression provided extra information that was useful in distinguishing oocyte stages. Comparing transcriptomes of the oocytes at the GV stage and the MII stage, in addition to identification of differentially expressed genes (DEGs), we detected many differentially expressed transcripts (DETs), some of which came from genes that were not identified as DEGs. When breaking down the isoform-level changes into alternative RNA processing events, we found the main source of isoform composition changes was the alternative usage of polyadenylation sites. With detailed analysis focusing on the alternative usage of 3′-UTR isoforms, we identified, out of 3,810 tested genes, 512 (13.7%) exhibiting significant switches of 3′-UTR isoforms during the process of moues oocyte maturation. Altogether, our data and analyses suggest the importance of examining isoform abundance changes during oocyte maturation, and further investigation of the pervasive 3′-UTR isoform switches in the transition may deepen our understanding on the molecular mechanisms underlying mammalian early development.

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Improving the diversity of captured full-length isoforms using a normalized single-molecule RNA-sequencing method

Cited by 9 publications

References 48 publications

An improved repertoire of splicing variants and their potential roles in Arabidopsis photomorphogenic development

An improved repertoire of splicing variants and their potential roles in Arabidopsis photomorphogenic development

Identification of Human Global, Tissue and Within-Tissue Cell-Specific Stably Expressed Genes at Single-Cell Resolution

Pervasive 3′-UTR Isoform Switches During Mouse Oocyte Maturation

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