title: Local and global factors affecting RNA sequencing analysis.
abstract: 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 GC 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.
authors: Department of Obstetrics and Gynecology, CS Mott Center for Human Growth and Development, Wayne State University School of Medicine, Detroit, MI 48201, USA.
authors: Edward
authors: E
authors: Sendler
authors: Graham D
authors: GD
authors: Johnson
authors: Stephen A
authors: SA
authors: Krawetz
mesh_terms: D001482:Base Composition / Q000235:genetics; D001483:Base Sequence; D006801:Humans; D008297:Male; D008969:Molecular Sequence Data; D009690:Nucleic Acid Conformation; D012313:RNA / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D020133:Reverse Transcriptase Polymerase Chain Reaction; D017423:Sequence Analysis, RNA / Q000379:methods*
chemical_list: C034477:RNA primers; D012313:RNA