Next generation sequencing (NGS), often referred to as massively paralleled sequencing, is a collective group of methods characterized by their high sequencing throughput (12). Currently available NGS platforms include the Illumina HiSeq/MiSeq, Life Technologies Ion Torrent/Ion Proton, Life Technologies SOLiD, and Roche 454. In contrast to Sanger sequencing, which produces a single long (often >1 kb) read using dye terminator chemistry, NGS methods typically generate millions of short reads on the order of 50–250 bp using reversible sequencing chemistries (13). NGS methods have allowed for unprecedented discovery in cancer, including acute myeloid leukemia, lung cancer, and breast cancer, and are now being applied in the clinical setting for evaluation of cancer predisposition syndromes, developmental delay, and cancer prognosis (14–19). NGS may be used to generate whole genome data, generate exome data (all coding sequences in the genome), or target specific genes or loci of interest (20). While whole genome data is generally low coverage (8–30 × coverage) and suitable for the detection of constitutional variants, by targeting sequencing to specific genes or regions of interest, coverage may be increased to 1,000× or higher, permitting more sensitive evaluation of gene variants and subclonal populations in cancer (21).
