GeneRead QIAact BRCA 1/2 Panel

The GeneRead QIAact BRCA 1/2 panel comprises 253 pooled primer pairs custom designed to cover all coding regions of the BRCA 1 and 2 genes, including 20bp flanking regions in adjacent introns. Designed as an integrated part of the GeneReader NGS System, and incorporating QIAGEN’s pre-eminent QCI automated bioinformatics for analysis and interpretation, this panel enables clinical research insights into both breast and ovarian cancers from 40 ng of input material. Target enriched DNA with the GeneRead QIAact BRCA 1/2 panel is ready for fully automated library preparation on the QIAcube instrument in the next step of the workflow.

In verification studies conducted with FFPE tumor samples and high quality DNA extracted from Coriell immortalized tumor cell lines as starting material, the GeneRead QIAact BRCA 1/2 panel performed to a high level on the GeneReader NGS System. At a 5% variant allele frequency the workflow demonstrated 100% precision and sensitivity, successfully identifying all mutations present in the samples tested. A high degree of reproducibility was observed between sample batches, workflow operators and GeneReader systems.

DNA sequencing is a useful tool to detect genetic variation, including somatic mutations, SNPs and small insertions and deletions. Target enrichment technology enables NGS-platform users to sequence specific regions of interest instead of the entire genome, effectively increasing throughput with lowering cost.

The GeneRead QIAact BRCA 1/2 Panel uses multiplex-PCR-based target enrichment technology in combination with a sophisticated primer-design algorithm. This technique enables amplification and enrichment of target regions in the human genome for detection of genetic variation using NGS. Adjacent and potentially interacting primer pairs are separated into different pools for optimal performance.

The GeneRead QIAact BRCA 1/2 Panel is used in the Target Enrichment step as an integrated part of the highly automatable GeneReader NGS System workflow. An overview of this system can be seen in figure 1.

The GeneRead QIAact BRCA 1/2 Panel is a PCR amplicon-based assay designed to amplify all coding regions of the BRCA1 and BRCA2 genes.The sequences of the amplicons are determined and compared with the reference sequences for the BRCA1/2 genes.

In the first step of the GeneRead QIAact BRCA 1/2 Panel workflow (Target PCR), the full coding regions of the BRCA1/2 genes (including at least 20 nucleotides adjacent) are amplified in 4 separate multiplex PCR for each sample.

In the second step of the workflow (Sample Pooling and Purification), the purified PCR products for each sample (total of 4 reactions) are pooled before proceeding to bead purification.

Subsequently the resulting purified PCR products from each sample are subjected to library construction. The purified PCR products generated from a single sample are individually bar coded with GeneReader-compatible adapters at each end prior to a universal PCR amplification. The prepared libraries are quantified using the QIAxcel instrument and pooled in equimolar amounts. The pooled amplified libraries are subjected to clonal amplification by emulsion PCR before sequencing on the GeneReader platform. Following sequencing, demultiplexed raw data files (FASTQ files) are imported to the QCI-A software for deeper analysis to identify variant positions compared with the reference sequences for the BRCA1/2 genes.

To ensure good quality results, in-process control criteria are used throughout the workflow, as outlined in the GeneRead QIAact BRCA 1/2 Panel Handbook . These criteria allow validation of the different steps of the workflow to identify samples that give poor sequencing results.

For target enrichment prior to next-generation sequencing (NGS) applications that use the QIAGEN GeneReader instrument.