Methods for in‐depth genome‐wide characterization of transcriptomes and quantification of transcript levels using various
microarray and next‐generation sequencing technologies 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 prior to measurements. This step has been shown to introduce many
biases and artifacts. In order to best characterize the ‘true’ transcriptome, the single‐molecule direct RNA sequencing (DRS)
technology was developed. This review focuses on the underlying principles behind the DRS, sample preparation steps, and the
current and novel avenues of research and applications DRS offers. WIREs RNA 2011 2 565–570 DOI: 10.1002/wrna.84
Figure 1.
Illustration of direct RNA sequencing sequencing preparation procedure. Poly-adenylated and 3′ blocked RNA is captured on surfaces containing covalently bound poly(dT) oligonucleotide (3′ end of the poly(dT) oligonucleotide faces up). A fill and lock step is performed, where the fill step is performed with natural thymidine and polymerase, and a lock step is performed with fluorescently labeled A-, C-, and G-Virtual-Terminator nucleotides and polymerase. These steps correct any misalignments that may be present in polyA and polyT duplexes and ensure that the sequencing starts in the RNA template rather than the poly-adenylated tail. Imaging is performed to locate the positions of the templates.
Diagram of single-molecule sequencing instrument optics. A 635-nm laser is used to illuminate the surface through the objective lens using total internal reflection. This generates an evanescent wave that results in a restricted excitation field, important for the reduction of background fluorescence. Fluorescent single molecules within the excitation field on the flow cell surface emit light, which is captured by the objective lens and detected by the charge-coupled device camera.
Professor David H Bechhofer joined Mount Sinai in 1986, after receiving his PhD from Columbia University in 1984 and doing postdoctoral work at the Public Health Research Institute of New York (moved since to UMDNJ in Newark, NJ). He is now Professor of Medical Education, and Professor of Pharmacology and Systems Therapeutics. His laboratory has been funded by the NIH since 1987, and he has served several times on the NIH Microbial Genetics Study Section as an ad hoc reviewer.
Professor Bechhofer’s current research interests focus on Prokaryotic mRNA decay and stable RNA processing. In particular he and his team study the mechanism of mRNA decay in the Gram-positive bacterium Bacillus subtilis. In early studies, the group showed that the 5’ end of a message is important in determining mRNA half-life, and they are now investigating the specific sites on an mRNA where decay begins.
