RNA-PET is a paired end tag (PET) sequencing method for full-length mRNA transcripts analysis using the next generation sequencer platforms such as Illumina GA and SOLiD. Unlike RNA-Seq method that sequences randomly sheared shotgun RNA short fragments, RNA-PET captures and sequences the 5′ and 3′ end tags of full-length cDNA fragments of all expressed genes in a biological sample. When mapped to reference genome, RNA-PET sequences can demarcate the boundaries of transcription units genome-wide, in addition to its ability to quantify the transcription level of each expression genes. Furthermore, the unique feature of RNA-PET is to identify fusion transcripts. Therefore, RNA-PET has been regarded as the best PET for genome annotation (1). Here in this chapter, we describe the details of the RNA-PET protocol and discuss the critical issues.
Series: Methods in Molecular Biology | Volume: 809 | Year: 2012 | Page Range: 535-562 | DOI: 10.1007/978-1-61779-376-9_35
Key Words: NGS - PET sequencing - Full length mRNA - Gene expression - Illumina GA - SOLiD - Transcriptome - RNA boundary - Splice variants - Fusion transcripts - EcoP15I