AbstractWhole-exome sequencing (WES) is a useful method to identify disease-causing mutations, however, often no candidate mutations are identified using commonly available targeted probe sets. In a recent analysis, we also could not find candidate mutations for 20.9% (9/43) of our pedigrees with congenital neurological disorder using pre-designed capture probes (SureSelect V4 or V5). One possible cause for this lack of candidates is that standard WES cannot sequence all protein-coding sequences (CDS) due to capture probe design and regions of low coverage, which account for approximately 10% of all CDS regions. In this study, we combined a selective circularization-based target enrichment method (HaloPlex) with a hybrid capture method (SureSelect V5; WES) and achieved a more complete coverage of CDS regions (~97% of all CDS). We applied this approach to 7 (SureSelect V5) out of 9 pedigrees with no candidates through standard WES analysis and identified novel pathogenic mutations in one pedigree. The application of this effective combination of targeted enrichment methodologies can be expected to aid in the identification of novel pathogenic mutations previously missed by standard WES analysis.