Whole transcriptome analysis: what are we still missing?

New technologies such as tag-based sequencing and tiling arrays have provided unique insights into the transcriptional output of cells. Many new RNA classes have been uncovered in the past decade, despite limitations in current technologies. Even as the repertoire of known functional elements of the transcriptome increases and contemporary technologies become mainstream, inadequacies in conventional protocols for library preparation, sequencing and mapping continue to hamper revelation of the entire transcriptome of cells. In this article, we review current protocols and outline their deficiencies. We also provide our view on what we may be overlooking in the transcriptome, despite exhaustive investigations, and indicate future areas of technological development and research.