GeneRead QIAact Lung UMI Panels

DNA sequencing is a useful tool to detect genetic variations, including somatic mutations, single nucleotide variants (SNVs), copy number variation (CNVs), andsmall insertions and deletions (InDels) from DNA, and fusions and exon skipping events from RNA.Targeted enrichment technology enables NGS platform users to sequence specific regions of interest instead of the entire genome or transcriptomeand effectively increase throughput with lower cost.The GeneRead QIAact Lung DNA UMIand Lung RNA FUsion UMIpanels are intended to be used either separately, or in combination to form the QIAact Lung All-in-One UMIPanel (instructions for use can be found in the handbook titled “GeneRead QIAact Lung All-in-One UMIPanel Assay) to offer a high level of assay flexibility. The Lung All-in-OneUMI Assay targets lung cancer-relevant mutations (SNVs, InDels, CNVs and fusions), therefore reducing the need for multiple tests to achieve a comprehensive view of the mutational landscape of a single lung cancer sample. The panels have also been optimized in combination with a specially formulated enrichment chemistry to achieve highly efficient enrichment on both regular and GC-rich regions at high multiplex levels.Existing target enrichment methods, library preparation and sequencing steps all utilize DNA polymerase and amplification processes, introducing substantial bias and artifacts. These biases and artifacts lead to background artefactual errors that greatly limit the detection of true low-frequency variants in heterogeneous samples such as tumors. The GeneRead QIAact Lung UMI Panels integrate unique molecular index (UMI) technology and a gene-specific, primer-based target enrichment process to enable sensitive variant detection of targeted regions by NGS on the GeneReader NGS System. The use of UMI technology results in higher confidence in variant calling, which is reflected in the high Q scores obtained.

The GeneRead QIAact Lung portfolio comprises:

• GeneRead QIAact Lung DNA Panel UMIKit (cat no. 181931) is designed to enrich selected genes and regions using 40 to 100 ng of DNA.
• GeneRead QIAact Lung RNA Fusion UMI Panel Kit (cat no. 181936) is designed to enrich selected fusion targets using 100 ng of total RNA.
• GeneRead QIAact Lung All-in-One UMI Assay which combines the GeneRead QIAact Lung DNA and Lung RNA Panel Kits (cat no. 181931 and 181936).

GeneRead QIAact Lung DNA UMI Panel

Genomic DNA samples are first fragmented, end-repaired and A-tailed within a single, controlled multi-enzyme reaction. The prepared DNA fragments are then ligated at the 5’ ends to a sequencing platform-specific adapter containing a UMI and a sample-specific bar code. Ligated DNA molecules are subject to limited cycles of target enrichment PCR. 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. Three DNA cleanup steps are required during this protocol for the removal of buffer and enzymes that might inhibit downstream library processing:

• A first cleanup procedure takes place after the adapter ligation
• A second cleanup procedure takes place after target enrichment PCR
• A final cleanup procedure is performed after the universal PCR step

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. Note: The GeneRead QIAact Cleanup Kit (cat no. 185446) is currently intend for us with just the GeneRead QIAact Lung DNA UMIPanel.

GeneRead QIAact Lung RNA Fusion UMIPanel

Total RNA is first reverse-transcribed to first strand cDNA. A separate, second strand synthesis is used to generate double strand (ds)-cDNA. This (ds)-cDNA is then end-repaired and A-tailed in a single tube protocol. The prepared (ds)-cDNAs are then ligated at the 5’ ends to a sequencing platform-specific adapter containing UMI and sample specific bar code. Ligated (ds)-cDNA molecules are subject to limited cycles of target enrichment PCR. 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.

GeneRead QIAact Lung All-in-One UMI Assay

Thisassay combines elements ofboth the GeneRead QIAact Lung DNA UMI Panel and GeneRead QIAact Lung RNA Fusion UMIPanel Kit protocols.Full detailscan be found in the GeneRead QIAact Lung All-in-OneUMI Assayhandbook.

Data analysis

Once the library is sequenced, results can be analyzed using the relevant GeneRead QIAact Lung assay workflow, which will automatically perform all steps necessary to generate a 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 RNA for digital sequencing as part of the GeneReader NGS System workflow.