Despite this progress, a complete understanding of the extent of intergenic transcription and the identity of these transcripts has remained elusive. The first attempts to analyze the extent and nature of intergenic transcription utilized tiling array technology [2]–[5]. These studies suggested that intergenic transcription is pervasive, but concerns about cross-hybridization have fueled a debate about the data [9]–[12]. Furthermore, in order to avoid technical difficulties associated with analyzing repeat sequence using tiling arrays, the studies were restricted to evaluating less than half of the genome. More recently, a few studies have focused on evaluating the extent of intergenic transcription using sequencing-based approaches, but with the exception of the recently published ENCODE project results [13], [14], these studies have thus far been limited to very narrow preselected regions of the genome and a small number of tissues [6], [7]. Overcoming these prior shortcomings, the ENCODE project used RNA-seq analysis in combination with other technologies to profile 15 human cell lines, providing evidence for transcription across 83.7% of the human genome and firmly establishing the reality of pervasive transcription [14].
