HaloPlex Target Enrichment Kit for Custom Targeted Sequencing

Protocol Overview

The protocol is lengthy and required two dedicated days as it includes a 16hr hybridization step. Initially, the DNA must be accurately quantified and diluted to the optimal concentration depending on the starting material (FF/FFPE). Samples are initially digested using 16 supplied restriction enzymes to create a library of gDNA restriction fragments.The results of the digestion need to be verified using either a Bioanalyzer, TapeStation or standard gel electrophoresis. Next, the collection of gDNA restriction fragments is hybridized to the HaloPlex probe capture library. The duration of thehybridization is determined by the probe density of the design, but could be up to 16hrs. During the hybridization, Illumina sequencing motifs including index sequences are incorporated into the targeted fragments. Following hybridisation, circularized target DNA-probe hybrids, containing biotin, are captured on streptavidin beads and then DNA ligase is added to the capture reaction to close nicks in the circularized probe-target DNA hybrids. The captured DNA is then eluted with NaOH and PCR is performed in order to amplify the captured target libraries. Finally, the amplified target libraries are purified and enrichment and quantity of the target DNA in each library sample is verified using the Bioanalyzer or the TapeStation.