The GeneRead QIAact AIT DNA UMI Kit is provided as two primer mix tubes, with approximately 500 primers per tube. The kit is designed to enrich specific target regions in select genes (AKT1, ALK1, BRAF, CTNNB1, DDR2, EGFR, ERBB2, ERBB3, ERBB4, ESR1, FBXW7, FGFR1, FGFR2, FGFR3, FLT3, GNA11, GNAQ, HRAS, KIT, KRAS, MAP2K1, MAP2K2, MET, NOTCH1, NRAS, PDGFRA, PIK3CA, RAF1, SMAD4, STK11) using 40-160 ng of DNA.
Genomic DNA samples are first fragmented, end-repaired and A-tailed using a single, controlled multi-enzyme reaction. The prepared DNA fragments are then ligated at their 5’ ends to a GeneReader specific adapter containing a UMI and a 9 base-pair (bp) sample-specific bar code.
Ligated DNA molecules are subject to limited cycles of target enrichment PCR, with gene-specific primers targeting hot spot regions paired with universal primers complimentary to an adapter sequence. Gene specific primers are tiled across the whole exon.This reaction ensures that intended targets and UMIs are enriched sufficiently to be represented in the final library. A universal PCR with GeneReader specific sequences is then carried out to amplify the targets and complete the library.
Once the library is sequenced, results can be analyzed using the GeneRead QIAact AIT DNA UMI Kit workflow, which will automatically perform all steps necessary to generate a DNA sequence variant report from your raw NGS data. All detected variants can be further interpreted by QIAGEN Clinical Insight (QCI) analysis.