SEQUENCING THE TRANSCRIPTOME IN TOTO

Since the sequencing of the mouse and human genomes, there has been a concerted effort to define their complete transcriptional output. EST, full length cDNA sequencing, and transcriptome annotation efforts by FANTOM, ENCODE and other consortia surveyed mammalian expression space, revealing that loci on average generate 6-10 transcripts. Alternative promoters, splicing and 3'UTRs are commonplace.

While these data have provided an excellent atlas of what can be generated from mammalian genomes, we have not had, until recently, the right genomic tools to place this transcriptional complexity into a biological context. Array based profiling has been an excellent tool for assessing overall gene activity, but lacks the sensitivity and resolution required to study complete transcriptome content.

RNA sequencing (RNAseq) has recently been demonstrated in several eukaryotic species and is redefining our understanding of mRNA transcriptome content and mRNA dynamics, all at a single nucleotide resolution. We have developed methods for performing multi-gigabase shotgun sequencing of human and mouse transcriptomes and have developed approaches to assess locus activity and demonstrated its improved sensitivity relative to the current "gold standard" array platforms. We also use RN Aseq to assess the expression levels of variant transcripts via diagnostic sequences. Thirdly, we are able to perform genome-wide transcriptome discovery. Finally we have also established approaches to identify alternations to the reference sequence content, allowing us to search for expressed polymorphisms, mutations or events such as RNA editing.

These data are combined with RNAseq surveys of other fractions of the transcriptome (i.e. small RNA and polysome-associated RNAs) to gain a fuller picture of coding and functional RNA content. This is being used to define, at unprecedented resolution, the transcriptional networks driving specific biological states.