Description
The QIAseq targeted DNA Panels have been developed as a complete Sample to Insight solution to enable digital DNA sequencing by utilizing molecular barcodes. Digital DNA sequencing is a unique approach to detect low-frequency variants with high confidence by overcoming the issues of PCR duplicates, false positives and library bias. Each panel is a one-box, NGS platform-agnostic solution that contains all the necessary components to construct libraries from enriched genomic targets. Primer design is based on QIAseq Enrichment Technology, in which each genomic target is enriched by one target-specific primer and one universal primer – a strategy that removes conventional two target-specific primer design restriction and reduces the amount of required primers. All primers required for a panel are pooled into an individual primer pool to reduce panel handling and the number of pools required for enrichment and library construction. Platform-specific indexes, which are contained in a separate box, allow the multiplexing of up to 384 samples per sequencing run.
Accuracy: Innovative digital sequencing (incorporating molecular barcodes) eliminates PCR duplication and amplification artifacts to detect low-frequency variants with high confidence (see figure Principle of molecular barcodes). Specificity: The unique combination of our proprietary primer design algorithm and rigorous testing of every primer assay guarantees high specificity and accurate results. Uniformity: The QIAseq Targeted DNA Panel workflow has been optimized to deliver highly uniform sequencing results, to ensure sequencing capacity is utilized very efficiently (see figure Uniformity). Sensitivity: Digital DNA sequencing approach is optimized to deliver high confidence in calling low-frequency DNA variants. Over 90% sensitivity for 1% NA12878 SNP and indel on typical coding region with false positive less than 15 per mega base region when variants are detected with tiled primer design to cover complete coding region of each gene. Universality: The chemistry used in the QIAseq targeted DNA panels and workflow is compatible with both regular and GC-rich genomic regions, allowing one to achieve 100% coverage of genes rich in GC content such as CEBPA and CCND1 (see figure: coverage of GC-rich genomic regions) Flexibility: The QIAseq targeted DNA panels offer a high degree of flexibility in content and sample multiplexing. Several cataloged panels have been developed for a wide range of applications. One can also build a custom panel for a specific content, or extend the contents of an existing cataloged panel. Up to 384 samples can be multiplexed using the QIAseq indexes.
PCR duplicates are a major issue in targeted DNA sequencing, since, through PCR amplification, they turn unique DNA molecules into identical DNA molecules that cannot be distinguished from each other. In addition, errors from PCR amplification and sequencing process may also be present in final reads that lead to false positive variants in sequencing results. This, in turn, results in the inability to confidently call DNA variants present at low frequencies in the starting DNA material. To overcome the issue of PCR duplicates and amplification artifacts, the QIAseq Targeted DNA Panels use digital sequencing by incorporating molecular barcodes into the starting DNA material before any amplification takes place, thereby preserving the uniqueness of the starting DNA molecules and overcoming the issues of PCR duplicates, false positives and library bias.
The entire workflow of the QIAseq targeted DNA panels to go from extracted DNA to sequencing-ready libraries can be completed in 9 hours (see figure Workflow). Extracted DNA is fragmented, genomic targets are molecularly barcoded and enriched, and libraries are constructed. Sequencing files can be fed into the QIAseq pipeline, a cloud-based data analysis pipeline, which will filter, map and align reads, as well as count unique molecular barcodes associated with targeted genomic regions, and call variants with a barcode-aware algorithm. This data can then be fed into IVA or QCI for interpretation.
