How sensitive is the rhAmpSeq™ CRISPR Analysis System?

With CRISPAltRations™, the software behind the rhAmpSeq CRISPR Analysis Tool, we analyzed the editing in three experimental replicates of 20 paired, treated, and control Cas9-edited loci. The treated samples had known indel editing levels (0.05, 0.10, 0.20, 0.50, 1.0, 2.0, and 3.4%), while the control samples had noise signals ranging from 0–0.4%. Using the Fisher’s Exact Test (with a threshold of p<0.5), we show that 95% sensitivity and specificity were obtained for detecting indels down to 0.5%. This analysis assumed that there were:

• more than 5000 paired-end reads in both the treatment and control samples
• ·normal levels of background indel noise (<0.4% indels)
• treatment sample loci with editing >0.5%
• libraries prepared with the rhAmpSeq CRISPR Library Kit and sequencing data analyzed by the rhAmpSeq CRISPR Analysis Tool (powered by CRISPAltRations®).