Sequencing of RNA (RNA-seq) is a recent technique that emerged shortly after next-generation sequencing (NGS) was invented approx. 10 years ago and since has revolutionized biological research in the 21st century. The major advance and basis of NGS is the application of sequencing-by-synthesis technology, which entails real-time monitoring of de novo DNA biosynthesis by imaging methods and reading out the sequence of newly synthesized DNA molecules upon iterative addition of the four different nucleotides. This is in contrast to “sequencing-after-synthesis”, which is based on the physical separation of differently sized DNA molecules generated by the chain-termination inhibitor method in polyacrylamide gels or by capillary electrophoresis after completion of the sequencing reaction (Sanger et al., 1977).
Most of the current sequencing-by-synthesis technologies are based on the immobilization of a denatured, single-stranded sequencing template on a surface, either a glass slide or nano-beads. Immobilization on a surface allows for repeated cycles of reagent delivery to the immobilized DNA molecule, which permits solid-phase oligo-nucleotide primer initiated synthesis of a new DNA strand, using repetitive and iterative cycles of addition of the nucleotides A, C, G, and T. High resolution imaging is used to detect the incorporation of the nucleotide, either during or after nucleotide incorporation, followed by iterative additional rounds of nucleotide incorporation. The sequence is then eventually deduced from the imaging data.
The first successful NGS approach that gained wide acceptance by the community was 454 sequencing, a massively-parallel pyrosequencing approach (Margulies et al., 2005). 454 sequencing is based on the detection of pyrophosphate released during de novo synthesis of a new DNA strand by DNA polymerase, which allows real-time measurements of DNA biosynthesis (Ronaghi, 1998). Pyrophosphate released during DNA synthesis is converted to ATP by the action of sulfurylase, followed by generation of a luminescent light signal from ATP, using firefly luciferase. (read more…)