Local and global factors affecting RNA sequencing analysis

被引:32
|
作者
Sendler, Edward [1 ]
Johnson, Graham D. [2 ]
Krawetz, Stephen A. [1 ,2 ]
机构
[1] Wayne State Univ, Sch Med, Dept Obstet & Gynecol, CS Mott Ctr Human Growth & Dev, Detroit, MI 48201 USA
[2] Wayne State Univ, Sch Med, CS Mott Ctr Human Growth & Dev, Ctr Mol Med & Genet, Detroit, MI 48201 USA
关键词
RNA; RNA sequencing; Sequence analysis; High-throughput RNA sequencing; SECONDARY STRUCTURE; EUKARYOTIC TRANSCRIPTOME; SEQ; QUANTIFICATION; ANNOTATION; BIASES;
D O I
10.1016/j.ab.2011.08.013
中图分类号
Q5 [生物化学];
学科分类号
071010 ; 081704 ;
摘要
High-throughput RNA sequencing (RNA-seq) continues to provide unparalleled insight into transcriptome complexity. Now the "gold standard" for assessing global transcript levels, RNA-seq is poised to revolutionize our understanding of transcription and posttranscriptional regulation of RNA. Despite significant advantages over prior experimental strategies, RNA-seq is not without pitfalls. We have identified a number of confounding factors that significantly affect sequencing coverage. These include regional CC content, preferential sites of fragmentation, and read "pile-up" due to primer affinity and transcript end effects. Independent of cell type and laboratory, when ignored, these factors can bias analyses. Understanding the underlying principles responsible for producing these artifacts is key to recognizing both their presence and how their effects may be controlled. Here we outline the causes of and strategies to avoid several previously unreported complicating factors common to RNA-seq experiments. (C) 2011 Elsevier Inc. All rights reserved.
引用
收藏
页码:317 / 322
页数:6
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