The tumor content of a biopsy and the average depth of coverage are two essential aspects when performing DNA sequencing using next-generation sequencing technologies. The heterogeneous nature of cancer necessitates the identification of distinct clonal cell populations to better understand and treat cancer. Deep sequencing enables researchers to identify these populations, but no consensus on an optimal depth exists for identifying clonal populations. Data from eight deep-sequenced oral squamous cell carcinoma biopsies obtained from three stage IV patients, with various degrees of tumor content, were used to randomly down sample the depth before being subjected to cluster analysis. An increase in coverage resulted in an increase in resolution for clusters of mutations, enabling the identification of distinct clonal cell populations and clonal events. From a depth of 800×, limited gain in resolution can be achieved; and from a depth of 1200×, the resolution stabilizes. Overall, researchers should aim for an average depth of 1000× to 1200× when performing deep sequencing. The tumor content will, however, dictate the resolution and fidelity of the analysis, as an increase in tumor complexity increases the need for higher tumor content.