TERA-Seq: true end-to-end sequencing of native RNA molecules for transcriptome characterization

被引:24
|
作者
Ibrahim, Fadia [1 ,3 ]
Oppelt, Jan [1 ]
Maragkakis, Manolis [2 ]
Mourelatos, Zissimos [1 ]
机构
[1] Univ Penn, Dept Pathol & Lab Med, Div Neuropathol, Perelman Sch Med, Philadelphia, PA 19104 USA
[2] NIA, Lab Genet & Genom, Intramural Res Program, NIH, Baltimore, MD 21224 USA
[3] Thomas Jefferson Univ, Sidney Kimmel Med Coll, Dept Biochem & Mol Biol, Philadelphia, PA 19107 USA
基金
美国国家卫生研究院;
关键词
MESSENGER-RNA; POLY(A) TAIL; GENE-EXPRESSION; DNA ELEMENTS; DECAY; IDENTIFICATION; REVEALS; LENGTH; ENCYCLOPEDIA; TRANSLATION;
D O I
10.1093/nar/gkab713
中图分类号
Q5 [生物化学]; Q7 [分子生物学];
学科分类号
071010 ; 081704 ;
摘要
Direct sequencing of single, native RNA molecules through nanopores has a strong potential to transform research in all aspects of RNA biology and clinical diagnostics. The existing platform from Oxford Nanopore Technologies is unable to sequence the very 5' ends of RNAs and is limited to polyadenylated molecules. Here, we develop True End-to-end RNA Sequencing (TERA-Seq), a platform that addresses these limitations, permitting more thorough transcriptome characterization. TERA-Seq describes both poly- and non-polyadenylated RNA molecules and accurately identifies their native 5' and 3' ends by ligating uniquely designed adapters that are sequenced along with the transcript. We find that capped, full-length mRNAs in human cells show marked variation of poly(A) tail lengths at the single molecule level. We report prevalent capping downstream of canonical transcriptional start sites in otherwise fully spliced and polyadenylated molecules. We reveal RNA processing and decay at single molecule level and find that mRNAs decay cotranslationally, often from their 5' ends, while frequently retaining poly(A) tails. TERA-Seq will prove useful in many applications where true end-to-end direct sequencing of single, native RNA molecules and their isoforms is desirable.
引用
收藏
页数:18
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