Targeted DNA

QIAseq Targeted DNA Panels integrate molecular barcode technology into a highly multiplexed, PCR-based, target enrichment process, enabling accurate variant calling at very low frequencies. During library preparation of the samples with a QIAseq Targeted DNA Panel, a Unique Molecular Index (UMI) and a common sequence prefix are added to each read. The resulting barcoded molecules are then amplified by PCR. After amplification, with different UMIs represent different original molecules, while reads with the same UMI result from amplification of the same original molecule. Target quantification is thus more accurate if the number of UMIs is counted rather than the number of reads.

The first steps in the QIAseq Targeted DNA Panel Analysis ready-to-use workflows involve trimming remaining PCR adapters, the UMI and the common sequence prefix while retaining the UMI barcoding information as an annotation on the read. This is followed by mapping the trimmed reads to the human reference sequence. After mapping, the Create UMI Reads from Grouped Reads tool generates a single consensus read, called a "UMI read", from reads with the same UMI.

The workflow then removes ligation artifacts from the read mapping. Next, the Indels and Structural Variants detection step generates a guidance track used for improving the mapping with the Local Realignment tool. Variants are then detected on the UMI reads using the Low Frequency Variant Detection tool for somatic workflows, and the Fixed Ploidy Variant Detection tool for germline applications. Finally, a series of filtering steps removes variants likely to be artifacts. The final output of the workflow includes, among other items, a list of filtered variants, including some present at very low frequency in the original dataset.