Variant interpretation depends on accurate annotations using biologically relevant transcripts. We have developed a systematic strategy for designating primary transcripts, and applied it to 109 hearing loss-associated genes that were divided into 3 categories. Category 1 genes (n=38) had a single transcript, Category 2 genes (n=32) had multiple transcripts, but a single transcript was sufficient to represent all exons, and Category 3 genes (n=38) had multiple transcripts with unique exons. Transcripts were curated with respect to gene expression reported in the literature and the Genotype-Tissue Expression Project. In addition, high frequency loss of function variants in the Genome Aggregation Database, and disease-causing variants in ClinVar and the Human Gene Mutation Database across the 109 genes were queried. These data were used to classify exons as "clinically relevant", "uncertain significance", or "clinically insignificant". Interestingly, 7% of all exons, containing >124 "clinically significant" variants, were of “uncertain significance”. Finally, we used exon-level next generation sequencing quality metrics generated at two clinical labs, and identified a total of 43 technically challenging exons in 20 different genes that had inadequate coverage and/or homology issues which might lead to false variant calls. We have demonstrated that transcript analysis plays a critical role in accurate clinical variant interpretation.