For clonal amplification of DNA libraries for next-generation sequencing (NGS) applications using the QIAGEN GeneRead QIAcube instrument
Generation of sequencing-ready amplified libraries
Multiplex clonal amplification: each library pool can include up to 12 uniquely bar coded DNA library samples
Automated on the GeneRead QIAcube
Proven performance with the GeneReader NGS System
Next-generation sequencing (NGS) is a driving force for numerous applications, including cancer research. After the completion of library preparation using the GeneRead DNA Library Q Kit, the GeneRead Clonal Amp Q Kit is used to prepare sequencing templates using clonal amplification.
Clonal amplification of 4 library pools; Reagents and Buffers for library concentration normalization and pooling, emulsion making, breaking and pooling, and library enrichment
The GeneRead Clonal Amp Q Kit is for Research Use Only. Not for use in diagnostic procedures.
Please note: The GeneReader NGS System is currently only available with proprietary new sequencing chemistry in the US. Legacy sequencing chemistry is only available ex-US.
The GeneRead Clonal Amp Q Kit enables processing of DNA library samples and pooling of multiple DNA library samples that have been uniquely bar coded during the library preparation process. The clonal amplification workflow is comprised of emulsion making, emulsion PCR, emulsion breaking and the enrichment of live beads. At the completion of the entire clonal amplification workflow, the DNA library sample/pools will have been clonally amplified and, after enrichment, are ready for sequencing.
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.
The GeneRead Clonal Amp Q Kit is designed for use in the GeneReader NGS System workflow.