Approaches to characterize heterogeneity. Tumor bulk sequencing: massively parallel sequencing of millions of tumor cells can be employed to assess the allele frequencies of mutations. Using statistical methods, the clonal frequencies of these mutations can be inferred. Detection of ultra-rare mutations: mutations that are present in rare populations of cancer cells (that is, comprise <1% of the tumor cell population) can be identified using high-fidelity sequencing, such as allele-specific tagging of DNA molecules, such that only alterations found on both strands are defined as mutations. Single-molecule sequencing: DNA is extracted from tumor cells and sequenced on a single-molecule sequencing platform (for instance, the Pacific Bioscience RS system). Single-cell sequencing: tumors are dissociated into single cells. DNA from single cells is amplified and sequenced using massively parallel sequencing to genotype individual cells. In situ topological genotyping: DNA or mRNA is amplified in situ on histological sections of the tumor, allowing for the genotyping of the cancer cells within the tumor without losing anatomical and histological information.