Accurate expression quantification from nanopore direct RNA sequencing with NanoCount.
Accurate expression quantification from nanopore direct RNA sequencing with NanoCount.
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DOI：
10.1093/nar/gkab1129
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发表时间：
2022-02-28
影响因子：
14.9
通讯作者：
Clark MB
中科院分区：
文献类型：
--
作者：
Gleeson J;Leger A;Prawer YDJ;Lane TA;Harrison PJ;Haerty W;Clark MB
准确地量化基因和异构体的表达变化对于了解细胞功能、分化和疾病是至关重要的。使用长读直接RNA测序(DRS)对全长天然RNA进行测序，有可能克服需要RNA片断、cDNA合成或PCR的短读和长读测序方法的许多局限性。然而，缺乏专门为DRS设计的工具，并且其识别复杂生物体中差异表达的能力特征不佳。我们开发了NanoCount来快速、准确地定量DRS中的转录异构体，并证明了它的性能优于类似方法。使用合成对照和人SH-SY5Y细胞分化为神经元样细胞，我们证明了DRS准确地量化了RNA的表达，并识别了基因和异构体的差异表达。在未分化和分化的SH-SY5Y细胞和分化程度不同的样本之间检测到231个基因、333个异构体和27个异构体开关在基因和异构体水平上的差异表达。神经元样细胞中上调的基因与神经发生有关。对DRS发现的数千种新的异构体进行NanoCount量化，同样能够识别它们的差异表达。我们的结果证明了增强的DRS异构体的纳米计数，并建立了DRS的能力，以确定生物相关的差异表达的基因和异构体。
Accurately quantifying gene and isoform expression changes is essential to understanding cell functions, differentiation and disease. Sequencing full-length native RNAs using long-read direct RNA sequencing (DRS) has the potential to overcome many limitations of short and long-read sequencing methods that require RNA fragmentation, cDNA synthesis or PCR. However, there are a lack of tools specifically designed for DRS and its ability to identify differential expression in complex organisms is poorly characterised. We developed NanoCount for fast, accurate transcript isoform quantification in DRS and demonstrate it outperforms similar methods. Using synthetic controls and human SH-SY5Y cell differentiation into neuron-like cells, we show that DRS accurately quantifies RNA expression and identifies differential expression of genes and isoforms. Differential expression of 231 genes, 333 isoforms, plus 27 isoform switches were detected between undifferentiated and differentiated SH-SY5Y cells and samples clustered by differentiation state at the gene and isoform level. Genes upregulated in neuron-like cells were associated with neurogenesis. NanoCount quantification of thousands of novel isoforms discovered with DRS likewise enabled identification of their differential expression. Our results demonstrate enhanced DRS isoform quantification with NanoCount and establish the ability of DRS to identify biologically relevant differential expression of genes and isoforms.
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影响因子：
14.9
作者：
Frankish A;Diekhans M;Ferreira AM;Johnson R;Jungreis I;Loveland J;Mudge JM;Sisu C;Wright J;Armstrong J;Barnes I;Berry A;Bignell A;Carbonell Sala S;Chrast J;Cunningham F;Di Domenico T;Donaldson S;Fiddes IT;García Girón C;Gonzalez JM;Grego T;Hardy M;Hourlier T;Hunt T;Izuogu OG;Lagarde J;Martin FJ;Martínez L;Mohanan S;Muir P;Navarro FCP;Parker A;Pei B;Pozo F;Ruffier M;Schmitt BM;Stapleton E;Suner MM;Sycheva I;Uszczynska-Ratajczak B;Xu J;Yates A;Zerbino D;Zhang Y;Aken B;Choudhary JS;Gerstein M;Guigó R;Hubbard TJP;Kellis M;Paten B;Reymond A;Tress ML;Flicek P
通讯作者：
Flicek P
影响因子：
7
作者：
Li, Runsheng;Ren, Xiaoliang;Zhao, Zhongying
通讯作者：
Zhao, Zhongying
DOI：
10.1261/rna.078703.121
发表时间：
2022-03
期刊：
RNA (New York, N.Y.)
影响因子：
--
作者：
Mulroney L;Wulf MG;Schildkraut I;Tzertzinis G;Buswell J;Jain M;Olsen H;Diekhans M;Corrêa IR Jr;Akeson M;Ettwiller L
通讯作者：
Ettwiller L
影响因子：
5.3
作者：
Hahm, KM;Sum, EYM;Orkin, SH
通讯作者：
Orkin, SH
影响因子：
16.6
作者：
Liu, Huanle;Begik, Oguzhan;Novoa, Eva Maria
通讯作者：
Novoa, Eva Maria