therascreen BRCA 1-2 NGS FFPE Kit
For qualitative detection of variants within the BRCA1 and BRCA2 genes
The therascreen BRCA 1-2 NGS FFPE Kit aids classification of ovarian cancers by identifying variants in human BRCA1 and BRCA2 genes using DNA derived from formalin-fixed paraffin-embedded (FFPE) ovarian tumor tissue.
The therascreen BRCA 1-2 NGS FFPE Kit is intended for in vitro diagnostic use in Europe.
To aid in the classification of ovarian cancers, the therascreen BRCA 1-2 NGS FFPE Kit provides an NGS-based a molecular diagnostic assay for use with FFPE ovarian tumor tissue. Variants in the BRCA1/2 genes are identified in coding regions as well as in at least 20 bp flanking each exon. Table 1 provides information about the 21,150 base pairs (bp) covered by the kit.
Table 1. Coverage information
* Normalization of the amplicon coverage for sequencing run was performed by multiplying the minimum coverage obtained by the ratio of 3 Gb (minimum sequencing data output recommended) to run sequencing output (Gb). In this case, the normalized minimum coverage is equivalent to 3 Gb of data per sequencing run.
† NA12878 is used as positive control in each sequencing run.
Over 200,000 women are diagnosed worldwide with primary ovarian cancer (OC) each year. OC has the highest mortality rate of all female gynecological cancers (1, 2). The vast majority of women are diagnosed at an advanced stage and the average five-year overall survival is nearly 45%. Approximately 15% of OC occurrences are attributable to germline mutations in the BRCA1 and BRCA2 genes and a smaller percentage is accounted for by other germline mutations. In contrast, a large fraction of OC cases can be attributed to a growing number of somatic aberrations (occurring only in the tumor mass itself) in critical tumor suppressor genes, including BRCA1/2 (3, 4).
As the majority of clinical OC specimens are FFPE tissue, subsequent analysis of DNA extracted from such FFPE tumor samples is qualitatively challenging. Unlike the clinically relevant mutation spectrum of other genes analyzed from FFPE tumor DNA (e.g., KRAS or EGFR), for which the distribution and number of mutations is small, thousands of clinically relevant variations in BRCA1/2 have been described. These BRCA1/2 variations are distributed widely throughout multiple, large coding regions and intron–exon boundaries (5). In addition, tumor samples are histologically heterogeneous (6), and tumor-specific DNA contains varying proportions of DNA from normal cells. Sanger DNA sequencing methods are typically not sensitive enough to detect low-level somatic changes and are difficult to scale for high-throughput applications. NGS offers a solution for these challenges, allowing comprehensive mutation screening of BRCA1/2 genes. This technology is explicitly supported by the Arbeitsgemeinschaft Gynäkologische Onkologie e.V. (AGO) for testing commonly mutated genes to better stratify patients (7).
The therascreen BRCA 1-2 NGS FFPE Kit provides a target-enrichment assay to prepare specific regions of interest for NGS. The assay allows qualitative detection of multiple nucleotide variants within the targeted DNA sequences purified from FFPE tumor samples. The kit provides 4 multiplexed primer mixes designed to amplify all coding regions of the BRCA1/2 genes including the 20 bp adjacent to each exon. Each run contains 10 samples, one positive control (NA12878) and one no-template control. After the amplification reaction is complete, the 4 reactions for each sample are pooled and purified. The amplicons are bar coded and amplified using a library preparation kit that is compatible with the Illumina MiSeq instrument. Afterwards, a first library quantification is performed before proceeding to library normalization and pooling. Subsequently, a second library quantification is performed to accurately normalize the pooled library. The pooled library is then ready for denaturation and sequencing with the Illumina MiSeqDx platform. Demultiplexed raw sequencing data (FastQ files) are imported to the CLC Biomedical Genomics Workbench software to identify genomic variations by comparing the data with the reference sequences for the BRCA1/2 genes.
The therascreen BRCA 1-2 NGS FFPE kit was developed for the QIAGEN universal workflow (see figure “Workflow therascreen BRCA 1-2 NGS FFPE Kit”), covering extraction to data analysis, in which sequencing is performed by the Illumina MiSeq Dx platform using Illumina MiSeq V2 cartridges.
The therascreen BRCA 1-2 NGS FFPE Kit enables identification of variants in the coding regions of the BRCA1/2 genes using DNA extracted from FFPE ovarian tumor tissue to aid in the classification of ovarian cancers.
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