The 5-step workflow is outlined below:
Library concentration normalization and pooling
- Manual normalization of the DNA library sample(s) concentration and pooling (if multiplexing is to be performed) are performed.
- The emulsion PCR beads are denatured in preparation for binding to the DNA library pool to eliminate bead clumping.
- Automated emulsion droplet formation is carried out on the GeneRead QIAcube.
- Upon completion of emulsion making, the PCR plate containing the emulsions is removed from the GeneRead QIAcube workdeck and manually loaded onto a stand-alone thermal cycler for clonal amplification.
Breaking and pooling
- The PCR plate containing the clonally amplified emulsions is loaded back onto the GeneRead QIAcube for the automated processes of pooling, breaking and cleaning of the emulsified library pools.
- Super A Beads are manually washed prior to the enrichment of live beads.
- Automated enrichment of live beads is conducted on the GeneRead QIAcube.
- Enriched beads are recovered and manually transferred from the GeneRead QIAcube into sample tubes.
Manual determination of bead concentration using OD
- Following the clonal amplification workflow, a standard curve is created using Primer Loaded PCR Beads that have not been through the clonal amplification process.
- The optical density of the enriched beads is measured so that the concentration can be extrapolated using the standard curve.
- Enriched beads (clonally amplified and enriched library pools) are then ready for sequencing.