Next Generation Sequencing (NGS)

NGS enables indel detection in both mixed populations and clonal cell lines. Its ability to process a large number of samples (samples are pooled together for a high-throughput sequencing run) and simultaneously screen off-target changes makes it a popular choice among researchers. In addition, as in all sequencing approaches, the nature and diversity of mutations are revealed

The data obtained from targeted amplicon sequencing is highly sensitive with detection levels as low as 0.01% (Hendel et al. 2015). This means that the researcher can be relatively certain that their samples do not contain off-target mutations if they are undetectable using this technique.

Besides being able to detect if all alleles of a gene were correctly edited, information on indel location and whether a cell population is truly monoclonal can be derived from NGS data.

Hendel, Ayal, et al. “Quantifying on-and off-target genome editing.” Trends in Biotechnology 33:2 (2015)