Deep Sequencing

Organizer: Eric Lai, Memorial Sloan Kettering Cancer Center

The goal of this group is to provide a forum for scientists engaged in research into the biology, biochemistry, and applications of RNAi silencing to discuss advances in this exciting new field and to promote interinstitutional and interdisciplinary dialogue.

New DNA sequencing platforms deliver several orders of magnitude more sequence than is possible with the traditional Sanger method, opening up new possibilities for the analysis of small RNA populations. This meeting will highlight "deep sequencing" projects in plants and animals, and the insights they reveal about gene regulation, genome structure, and genome evolution.

Agenda

5:00-5:10 pm Welcome and Introduction
5:10-5:50 pm Graham Ruby
5:50-6:30 pm Katsutomo Okamura
6:30-7:10 pm Michael Snyder
7:10-7:30 pm Open Discussion

Abstracts

The 21U-RNAs of Caenorhabditis Elegans
Graham Ruby, MD, Whitehead Institute for Biomedical Research, MIT

Non-coding RNAs comprise a wide variety of molecular species that can be categorized according to their intrinsic molecular structures and/or mechanisms of biogenesis. Deep sequencing of small (20-some nucleotide) RNAs from C. elegans revealed a new class of non-coding RNAs, the 21U-RNAs, with properties distinct from those of both microRNAs (miRNAs) and small interfering RNAs (siRNAs), the other two known classes of nematode small RNAs. The 21U-RNAs derive from over 10,000 individual loci, each of which is defined by a cis motif that appears sufficient for the production of a 21nt RNA and is conserved across nematodes. In contrast, the 21U-RNAs themselves are not conserved and share little sequence similarity with one another beyond their 5´ U. The pervasiveness and conservation of the cis motif presumably reflects its importance for producing these diverse, autonomously expressed, small RNAs (dasRNAs).

New Classes of Drosophila Small RNA Genes Revealed by Deep Sequencing
Katsutomo Okamura, MD, Memorial Sloan Kettering Cancer Center

Small RNA gene discovery was revolutionized by the recent application of "deep sequencing" techniques. In collaboration with the Bartel and Hannon groups, we used the 454 and Solexa platforms to analyze small RNA populations in Drosophila. In addition to refining and extending Drosophila miRNA annotations, these studies revealed new classes of small RNA-generating loci. We are currently studying the biogenesis and function of two types of small RNAs, mirtrons and endogenous siRNAs. The former are alternate precursors for miRNAs, while the latter are novel 21-22 nt RNAs that are defined by their dependence on Drosophila Dicer-2 and Ago-2.

Deep Sequencing for Genome Analysis
Michael Snyder, MD, Yale University

We have used NextGen sequencing technologies for a variety of applications for analyzing genomes.
1) 454/Roche technology was used to determine the DNA sequence of the genome of Acinetobacter baummannii, an important human pathogen.
2) 454/Roche technology was used to identify and analyze structural variation in humans.
3) Illumina/Solexa sequencing has been used to map transcription factor binding sites in metazoan genomes.
These and other applications will be presented at the meeting.