Single‐molecule direct RNA sequencing without cDNA synthesis

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.