Isolated DNA, as low as 20 ng, is enzymatically fragmented to generate small pieces of dsDNA. This is followed by the library construction step, in which IL-N7 adapters, molecular barcodes, and sample indexes are incorporated into DNA fragments generated in the previous step. Library fragments now serve as templates for target enrichment using single primer extension. In this step, targets are enriched using a single gene-specific primer and a universal forward primer. The final step is library amplification and sample indexing (for dual indexing) using the IL-S5 sample index primer and a universal primer.
The QIAseq DNA panels use a proprietary buffer mixture to efficiently sequence GC-rich regions within the genome. Two examples are shown here: CEBPA and CCND1. Complete coverage of exonic regions within those two genes is achieved.
A variant identified in a sample represents one of two events: a true or false variant. False variants can be introduced at any step during the workflow, including sequencing reactions. This results in the inability to accurately and confidently call rare variants (those present at 1% of the sample). Due to PCR duplicates generated in amplification steps, all DNA fragments look exactly the same, and there is no way to tell whether a specific DNA fragment is a unique DNA molecule or a duplicate of a DNA molecule. With molecular barcodes, since each unique DNA molecule is barcoded before any amplification takes place, unique DNA molecules are identified by their unique barcodes, and PCR duplicates carrying the same barcode are removed, thereby increasing the sensitivity of the panel.
The QIAseq targeted DNA panels deliver outstanding sequencing metrics. 6000 SNPs were enriched from 20 ng of NA12878 DNA. Library was constructed for sequencing on a MiSeq, with 4,000,000 reads generated. The panel achieved a uniformity of 99.5% at 0.2x of mean coverage, and 96% at 0.5x of mean coverage.