Variants in tumor DNA can result in heterogeneous alignment patterns in tumor samples. (a) The progression of tumor development illustrates how neighboring cells can have several different genomic sequences. The tumor cells (orange) originate with a genetic mutation in one cell and grow rapidly to outnumber the normal cells (blue). During the rapid growth of the tumor, additional genomic variants are created through further mutations (brown), resulting in a heterogeneous mix of normal and multiple tumor genomic sequences. (b) Reads sampled from the normal cells (blue) and two subpopulations of tumor cells (orange/brown) are aligned against the reference sequence. Soft-clipped portions are indicated by a dotted line border. A germline-acquired deletion is present in all of the sample, but deletions present in only the tumor cells result in some reads being aligned to the reference and others being soft-clipped. Disregarding the possibility of heterogeneous sequencing samples might result in these variants being missed.