Bead Capping Protocol

Both unamplified and amplified beads will have unextended forward ePCR primers. The 3’ ends of these primers, as well as the 3’ ends of the RDV segment of the template DNA, must be capped to prevent fluorescent probes from ligating to these ends and degrading the sequencing read accuracy. At the same time, we wish to attach amine functional groups to the template DNA for subsequent coupling to the aminosilanated flow cell coverslip. A mixture of two bridging oligos is added to a suspension of the enriched, ePCR beads. One of these oligos hybridizes to the forward (FDV) segment of both unextended and extended ePCR primers, while the other hybridizes to the RDV segment of template DNA. The two bridge oligos are each designed to overhang their hybridization targets. A tertiary ligation mixture is then prepared and added, all three components of which terminate in an amine group. Two of the amine-modified oligos hybridize to the overhanging bridge oligos, and ligate to the ends of the primers, with the two components accommodating both A-tailed and non-Atailed primers. The third, degenerate component caps other species that may be present, including incomplete extension products. The capped beads are then washed via magnetic separation, and are ready to be arrayed.