D.P.4 Comparison of commercially-available exome capture kits in the diagnosis of neuromuscular disorders

Next generation sequencing technologies present opportunities for improved clinical molecular diagnosis of neuromuscular disorders (NMD). We sought to determine the feasibility of using whole exome sequencing to detect mutations in 347 isoforms of 143 NMD genes listed in the World Muscle Society Muscle Gene Table (www.musclegenetable.org). We initially tested DNA samples from eight subjects with limb-girdle muscular dystrophies (LGMD) and known mutations, using exome capture with one of three commercially available kits. We detected the same mutations as gene-specific Sanger sequencing had previously detected in seven of eight, missing 1 MYOT mutation due to a lack of representation of this specific exon in the capture kit. This led us to compare commercially available exome capture kits for coverage of NMD genes, by both predicted and by empiric coverage, in normal control genomic DNA. Based on manufacturer targeted region files, the three kits each targeted 92–94% of the known NMD exons; however, our actual capture results demonstrated that at best ∼60% of these exons obtained 100% coverage. In our hands the best performing kit, Agilent SureSelect v3, captures an average of 92.7% of the bases in the NMD gene exons, but only 58% of the NMD isoforms had all exons captured at 100% (e.g., 42% of the NMD genes had at least one exon with one base insufficiently captured to make a genotyping call). Illumina TruSeq captured 89.2% of exons but only 36.5% of genes had 100% coverage, and Nimblegen v2 captured 90.5% of genes but only 46.3% genes had 100% sequence coverage. We predict that by utilizing all three kits, ∼70% of the known NMD isoforms would have sufficient sequence coverage to make a genotyping call for 100% of all exons. When considering adoption of this approach clinically, this is an important limitation to be aware of but continued improvements in commercially available capture kits should allow economical and rapid NMD gene sequence analysis.