During the past five years , new high-throughput DNA sequencing technologies have emerged; these technologies are collectively referred to as next generation sequencing (NGS). By virtue of sequencing clonally amplified DNA templates or single DNA molecules in a massively parallel fashion in a flow cell, NGS provides both qualitative and quantitative sequence data. This combination of information has made NGS the technology of choice for complex genetic analyses that were previously either technically infeasible or cost prohibitive. As a result , NGS has had a fundamental and broad impact on many facets of biomedical research. In contrast , the dissemination of NGS into the clinical diagnostic realm is in its early stages. Though NGS is powerful and can be envisioned to have multiple applications in clinical diagnostics , the technology is currently complex. Successful adoption of NGS into the clinical laboratory will require expertise in both molecular biology techniques and bioinformatics. The current report presents principles that underlie NGS including sequencing library preparation , sequencing chemistries, and an introduction to NGS data analysis. These concepts are subsequently further illustrated by showing representative results from a case study using NGS for targeted resequencing of genes implicated in hypertrophic cardiomyopathy.