Methods for in-depth characterization of transcriptomes and quantification of transcript levels have emerged as valuable tools for understanding cellular physiology and human disease biology, and have begun to be utilized in various clinical diagnostic applications.  Current methods, however, typically require RNA to be converted to complementary DNA (cDNA) via reverse transcription prior to measurements.  This step has been shown to introduce biases and artifacts that may interfere with both the proper characterization and quantification of transcripts. To enable virtually unbiased view and quantification of transcriptomes, Helicos has developed and commercialized the single molecule Direct RNA Sequencing (DRS^TM) technology. DRS^TM is the first and currently the only technology that can sequence RNA molecules directly in a massively-parallel manner without RNA conversion to cDNA or other biasing sample manipulations such as ligation and amplification. DRS^TM has been applied to all RNA analysis needs including, but not limited to:

·          Quantitative polyadenylation site mapping/Digital Gene Expression

·          Whole transcriptome analyses including splice form characterization

·          Formalin-fixed, paraffin-embedded RNA sample quantitation and characterization

·          Small RNAs/Noncoding RNAs

·          RNA Immunoprecipitation

·          Characterization of RNA samples with very limited, attomole-level quantities

·          Single step selection and sequencing of target RNA molecules

Recent publications featuring the DRS^TM technology include:

Direct RNA sequencing. Nature 461, 814-818.

Comprehensive Polyadenylation Site Maps in Yeast and Human Reveal Pervasive Alternative Polyadenylation. Cell 143, 1018-1029.

Direct RNA Sequencing. Experimental Medicine 28: 2574-2580.

Lkb1 regulates stem cell quiescence, energy metabolism, and cell survival in the hematopoietic system. Nature. 2010 Dec 2;468(7324):659-63.

Single-molecule direct RNA sequencing without cDNA synthesis. WIREs

RNA. 2011; 2(4):565-570. Epub 2011 Mar 17.

RNA sequencing: advances, challenges and opportunities. Nat Rev Genet. 2011 Feb;12(2):87-98. Epub 2010 Dec 30.

The Approach
For profiling of RNA species with natural poly-A tails, sample preparation steps is not needed. Isolated total RNA or cell lysates are directly introduced to the poly(dT)-coated flow cells, which enable capture and sequencing of polyA RNA species. For RNA species such as small RNAs which generally do not contain a natural polyA tail, polyA polymerase is used to generate a polyA tail before loading the sample to the flow cells for sequencing. For certain applications such as whole transcriptome profiling, RNA fragmentation may be needed. This fragmentation step can be performed with a variety of ways and is chosen by the user. For the single step target selection and sequencing application, no sample preparation steps are needed.