The QIAGEN GeneReader Platform uses a sequencing-by-synthesis (SBS) approach for deciphering the DNA sequence of specific gene targets. Each cycle of SBS involve 3 major steps: DNA extension, signal detection and cleavage.
During the extension step, the DNA polymerase incorporates reversible, terminated fluorescent labeled deoxyribonucleotide triphosphates (dNTPs) to the DNA template. The reversible terminators enable the addition of only one fluorescence-labeled nucleotide at a time to the growing strand of the DNA template. This step is followed by a signal detection step carried out by the GeneReader scanner and imaging system, where signal from each bead is recorded. The cycle is completed with a cleavage step, in which the fluorescence dyes and the terminators are cleaved from the incorporated nucleotides, allowing the next cycle of incorporation. This technique ensures highly accurate and cost-effective NGS.
DNA libraries are clonally amplified on beads using the GeneRead QIAcube to become a sequencing template. After hybridization of a sequencing primer the primer-template carrying beads are immobilized via direct bead-glass interaction to produce a high-density array on a GeneReader flow cell. To read out the content of templates on each bead, the array of fragments is first subjected to reagents containing uniquely engineered dNTPs, as described above These bases are incorporated by a modified DNA polymerase to the end of the growing strand of DNA in accordance with the base on the complementary strand. The array is subsequently scanned by a high-resolution digital camera and the fluorescent output of each of the four dye colors at each array position is measured and recorded. Finally, the array is exposed to cleavage chemistry to break off the fluorescent dyes and reversible terminators that will then allow additional bases to be added. This cycle is then repeated on the GeneReader.