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 post-transcriptional regulation of RNA.
To fully capitalize on the opportunities afforded by RNA-seq one must be aware of its limitations. Common pitfalls in sample preparation and analysis can bias the representation in a template dependent manner. Their effect on the alignment of sequencing reads to the genome skews both the inferred transcript structure as well as relative abundance.
The authors have identified a number of confounding factors that significantly impact sequencing coverage and outline the causes and strategies to avoid several previously unreported complicating factors common to RNA-seq experiments, including:
- regional GC-content
- preferential sites of fragmentation
- read “pile-up” due to primer affinity and transcript-end effects
Sendler E, Johnsonm GD, Krawet SA. (2011) Local and global factors affecting RNA-seq analysis. Anal Biochem [Epub ahead of print]. [abstract]