Why do I need to use next-generation sequencing (NGS) to analyze the results of the screen? How can I perform this analysis?
Why do I need to use next-generation sequencing (NGS) to analyze the results of the screen? How can I perform this analysis?
NGS provides an efficient method for quantifying and comparing the frequencies of sgRNAs encoded in the screened and control populations, which in turn allows researchers to identify which gene knockouts yielded phenotypes relevant to the screen.
Following the screen, gDNA is purified from the screened and control cell populations, and proviral sgRNA sequences are PCR amplified and incorporated into NGS libraries. Following sequencing, software packages such as MAGeCK or even Excel can be used to compare the frequencies of sequencing reads corresponding to each sgRNA in the respective populations.