GeneRead QIAact Lung UMI Panels

Lung cancers are characterized by abundant genetic diversity, typically requiring the application of multiple assays in order to achieve cancer research insights. The GeneRead QIAact Lung DNA UMI combined with the GeneRead QIAact RNA Fusion UMI Panel to become the Lung All-in-One UMI Assay, is the first targeted gene panel to consolidate all relevant mutations into a single next-generation sequencing (NGS) assay.
The selection of the QIAact Lung DNA UMI Panel’s 550 variants across 19 genes, and the 79 fusions in genes commonly affected by translocations covered by the QIAact Lung RNA Fusion UMI Panel, were guided by the Lung Cancer Alliance; an international team of lung cancer research specialists, to ensure an assay of unparalleled relevance. Incorporating QIAGEN’s Unique Molecular Index (UMI) technology, these panels offer more accurate quantification and detection of molecular variants on the GeneReader than ever before, from as little as 40 ng of input DNA or 100 ng of total RNA.
To further streamline the library preparation protocol, the GeneRead QIAact Panel Cleanup Kit offers an optimized protocol automated on the QIAcube, saving time and preventing handling errors for DNA library preparation. Designed to simplify the workflow, these kit contain all regents required for both the target enrichment and library preparation steps of the GeneReader NGS System workflow.
*Currently liquid biopsy workflow is only available for GeneRead QIAact Lung DNA UMI Panel. Protocol for RNA extracted from liquid biopsy samples coming soon.

DNA sequencing is a useful tool to detect genetic variations, including somatic mutations, single nucleotide variants (SNVs), copy number variation (CNVs), and small 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 transcriptome and effectively increase throughput with lower cost.
The GeneRead QIAact Lung DNA UMI and Lung RNA FUsion UMI panels are intended to be used either separately, or in combination to form the QIAact Lung All-in-One UMI Panel (instructions for use can be found in the handbook titled “GeneRead QIAact Lung All-in-One UMI Panel Assay) to offer a high level of assay flexibility. The Lung All-in-One UMI 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 UMI Kit (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 UMI Panel.

GeneRead QIAact Lung RNA Fusion UMI Panel

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

This assay combines elements of both the GeneRead QIAact Lung DNA UMI Panel and GeneRead QIAact Lung RNA Fusion UMI Panel Kit protocols. Full details can be found in the GeneRead QIAact Lung All-in-One UMI Assay handbook.

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.