Fig. 1.

DRS sample preparation. RNA species which contain a 3' poly-A tail require 3' end blocking as described. Other RNA species are enzymatically 3' polyadenylated and 3' blocked. The blocking step is performed to prevent “downward” nucleotide additions to the 3' end of the template during the sequencing process (details of the sequencing strategy and chemistry have been described previously (32)). Polyadenylated RNA is captured on the sequencing flow cell surfaces coated with poly(dT) oligonucleotides through hybridization. A “fill” step is performed with dTTP and polymerase, and then the templates are “locked” in position with fluorescently labeled proprietary Virtual Terminator™ (VT)-A, -C and -G sequencing nucleotide analogs. VT analogs are nucleotides used for sequencing, containing a fluores-cent dye and chemically cleavable groups that prevent the addition of another nucleotide. These “fill and lock” steps correct for any misalignments that may be present in poly-A/T duplexes, and ensure that the sequencing starts in the template rather than the poly-A tail.