With multigene panels becoming more accessible and cost effective for disease research, their use in the clinic is also becoming more accepted as an alternative to traditional gene by gene analysis for diagnostic use and disease risk assessment. However, in many cases, clinical guidelines for the use of these tests have yet to be fully established and technical questions addressing analytical performance, variant interpretation concordance, and yield of various classes of findings from panel testing still need to be addressed.
In this paper the authors compare NGS-based panel testing with traditional genetic testing for assessing risk of hereditary breast and ovarian cancer (HBOC) in a large population of over 1000 patients.
Lincoln et al. used an NGS-based gene panel covering 29 hereditary cancer genes to test 1062 patients previously indicated for HBOC risk assessment. Most of the samples had been subjected to traditional BRCA testing previously, while some had undergone other genetic tests or had high-quality reference data available. Reference samples containing known pathogenic variants were included as control specimens to measure coverage variability.
Targeted genes were captured from whole blood genomic DNA using SureSelect® probes (Agilent). xGen® Lockdown® Probes from IDT were used to rescue drop-out regions of SureSelect probe panels, thus providing more uniform target coverage. Sequencing was performed on the Illumina MiSeq® or HiSeq® 2500 to at least 450X average coverage of 2 x 150 reads; a minimum of 50X reads was required at every targeted position.
After alignment to the reference human genome, sequence variants were called using several established of algorithms. CNVs were called using read-depth analysis, and split-read analysis was performed to identify certain structural variants that can be overlooked using read-depth analysis and indel detection. The results were then compared with existing findings from traditional testing of the samples.
Results and conclusion
The researchers observed 100% analytical concordance of gene panel data with prior corresponding tests and were able to uncover previously unidentified pathogenic variants of apparent clinical relevance in some genes. While these results are specific to the gene panel, laboratory protocols, and bioinformatics analysis used, the authors suggest that NGS panel testing can serve as a viable replacement for traditional genetic tests in appropriate circumstances and can lead to potentially actionable findings that may be otherwise missed.