GeneRead QIAact Custom Panels

DNA sequencing is a useful tool to detect genetic variations, including somatic mutations, single nucleotide variants (SNVs), copy number variation (CNV) and small insertions and deletions (InDels), and sequencing of cDNA can be used to detect fusions.Targeted enrichment technology enables next-generation sequencing (NGS) platform users to sequence specific regions of interest instead of the entire genome and effectively increase throughput with lower cost. The GeneRead QIAact DNA Custom Panels are based on customer requirements, enabling you to specify the regions of specific interest to your research.Existing target enrichment methods, library preparation and sequencing steps all use DNA polymerase and amplification processesthat can introduce artifacts and substantial bias. These artifacts and biases lead to background artefactual errors that greatly limit the detection of true low-frequency variants in heterogeneous samples such as tumors. The GeneRead QIAact Custom Panels integrate unique molecular index (UMI) technology and a gene-specific, primer-based target-enrichment process to enable sensitive variant detection of targeted genomic regions by NGS on the GeneReader system. The use of UMI technology results in higher confidence variant calling, which is reflected in the high Q scores obtainable.

The GeneRead QIAact Custom Panels areprovided as two primer mix tubes, with a customer-specified number of primers per tube andare designed to enrich selected genes and regions using 40 ng of DNA or 100 ng of RNA.Genomic DNA samples are fragmented, end-repaired and A-tailed within a single, controlled multi-enzyme reaction. RNA samples are subjected to first-strand cDNA synthesis, reverse transcription and second-strand synthesis before end repair and A-tailing. The prepared double-stranded DNA fragments are then ligated at their 5’ ends to a specific adapter containing a UMI and a sample-specific bar code.Ligated DNA molecules are subject to limited cycles of target enrichment PCR, with gene-specific primers targeting genomic regions and a universal primer complimentary to an adapter sequence. This reaction ensures that intended targets and UMIs are enriched sufficiently to be represented in the final library. A universal PCR is then carried out to amplify the library and add GeneReader specific sequences which completes the library.Cleanup steps are required after the following proceduresfor the removal of buffer and enzymes that might inhibit downstream library processing:• Adapter ligation• Target enrichment PCR• Universal PCR Each of these cleanup steps is fully automatable on the QIAcube platform with the GeneRead QIAact Cleanup Kit (catalogue no. 185446) to reduce sample-processing time and risk of handling errors.Once the library is sequenced, results can be analyzed using the GeneRead QIAact Custom Panel 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.

For constructing targeted, molecularly bar-coded libraries from DNA and/or RNAfor digital sequencing as part of the GeneReader NGS System workflow.