Implementation of Targeted Next Generation Sequencing in Clinical Diagnostics

Accurate mutation detection is essential in clinical genetic diagnostics of monogenic hereditary diseases. Targeted next generation sequencing (NGS) provides a promising and cost-effective alternative to Sanger sequencing and MLPA analysis currently used in most diagnostic laboratories. One advantage of targeted NGS is that multiple disease-specific genes can easily be sequenced simultaneously, which is favorable in genetic heterogeneous diseases. Prior to implementation in our diagnostic setting, we aimed to assess the sensitivity and specificity of targeted NGS by sequencing a collection of mutation positive controls previously characterized by Sanger/MLPA analysis. Agilent SureSelect Target-Enrichment kits were used for capturing a set of genes associated with hereditary breast and ovarian cancer syndrome and a compilation of genes involved in multiple rare single gene disorders, respectively. For diagnostics, the sequencing coverage is essential, wherefore a minimum coverage of 30x per nucleotide in the coding regions was used as our primary quality criterion. For the majority of the included genes, we obtained adequate gene coverage, in which we were able to detect 100% of the known variants, including a 85 bp deletion and whole-exon duplications/deletions. We demonstrate that targeted NGS can replace Sanger/MLPA analysis in clinical diagnostics for the majority of genes without further optimization.