Nanopore long-read RNAseq reveals widespread transcriptional variation among the surface receptors of individual B cells.
Nanopore long-read RNAseq reveals widespread transcriptional variation among the surface receptors of individual B cells.
复制标题
DOI：
10.1038/ncomms16027
复制
发表时间：
2017-07-19
影响因子：
16.6
通讯作者：
Vollmers C
中科院分区：
文献类型：
--
作者：
Byrne A;Beaudin AE;Olsen HE;Jain M;Cole C;Palmer T;DuBois RM;Forsberg EC;Akeson M;Vollmers C
理解基因调控和功能需要一种全基因组方法，该方法能够在单细胞水平上同时获取基因表达水平和异构体多样性。短读长RNA测序在解析复杂异构体的能力上存在局限性，因为它无法对RNA分子的全长cDNA拷贝进行测序。在此，我们研究使用长读长单分子牛津纳米孔MinION测序仪进行的RNA测序是否能够在不牺牲准确的基因表达定量的情况下识别和定量复杂异构体。在对我们的方法进行基准测试之后，我们使用一种定制的多重策略分析单个小鼠B1a细胞。我们在这些B1a细胞中鉴定出数千个未注释的转录起始和终止位点，以及数百个可变剪接事件。我们还鉴定出在B1a细胞中表达的数百个基因呈现多种复杂异构体，包括几种B细胞特异性表面受体。我们的结果表明我们能够在单细胞水平上识别和定量复杂异构体。
短读长RNA测序在解析复杂转录异构体的能力上存在局限性，因为它无法对全长cDNA进行测序。在此作者使用牛津纳米孔MinION及其Mandalorion分析流程来测量B1a细胞中的复杂异构体。
Understanding gene regulation and function requires a genome-wide method capable of capturing both gene expression levels and isoform diversity at the single-cell level. Short-read RNAseq is limited in its ability to resolve complex isoforms because it fails to sequence full-length cDNA copies of RNA molecules. Here, we investigate whether RNAseq using the long-read single-molecule Oxford Nanopore MinION sequencer is able to identify and quantify complex isoforms without sacrificing accurate gene expression quantification. After benchmarking our approach, we analyse individual murine B1a cells using a custom multiplexing strategy. We identify thousands of unannotated transcription start and end sites, as well as hundreds of alternative splicing events in these B1a cells. We also identify hundreds of genes expressed across B1a cells that display multiple complex isoforms, including several B cell-specific surface receptors. Our results show that we can identify and quantify complex isoforms at the single cell level. Short-read RNA-seq is limited in its ability to resolve complex transcript isoforms since it cannot sequence full-length cDNA. Here the authors use Oxford Nanopore MinION and their Mandalorion analysis pipeline to measure complex isoforms in B1a cells.
登录
查看更多内容
影响因子：
9.2
作者：
Kilianski A;Haas JL;Corriveau EJ;Liem AT;Willis KL;Kadavy DR;Rosenzweig CN;Minot SS
通讯作者：
Minot SS
影响因子：
4.4
作者：
Karlsson K;Linnarsson S
通讯作者：
Linnarsson S
影响因子：
56.9
作者：
Femino, A;Fay, FS;Singer, RH
通讯作者：
Singer, RH
影响因子：
4.4
作者：
Dueck HR;Ai R;Camarena A;Ding B;Dominguez R;Evgrafov OV;Fan JB;Fisher SA;Herstein JS;Kim TK;Kim JM;Lin MY;Liu R;Mack WJ;McGroty S;Nguyen JD;Salathia N;Shallcross J;Souaiaia T;Spaethling JM;Walker CP;Wang J;Wang K;Wang W;Wildberg A;Zheng L;Chow RH;Eberwine J;Knowles JA;Zhang K;Kim J
通讯作者：
Kim J
影响因子：
78.5
作者：
Irish, JM;Kotecha, N;Nolan, GP
通讯作者：
Nolan, GP