Fig. 2: Optimized OliTag-seq Library Preparation via Triple Priming.
From: OliTag-seq enhances in cellulo detection of CRISPR-Cas9 off-targets
a Nested-PCR for gDNA Amplification: Genomic DNA, prepared with shearing and end-processing, is ligated to adaptors with unique molecular identifiers (UMIs). The initial PCR usesthree primers that anneal to the oligo tag and adaptor, creating a trio of product types. A second PCR stage amplifies these products to enrich flanking sequences around the dsODN insertion. The collective PCR output is then sequenced using 150 bp paired-end reads. b Enhanced Read Generation with Triple Priming: Triple priming in the first PCR markedly boosts read quantity for target sites compared to standard PCR methods, based on samples (n = 4 or 7 per site). These triple-priming results are normalized against a baseline established by double priming across two reactions. c Improved Off-Target Detection via Triple Priming: A single-reaction triple priming approach yields more detectable off-target sites, with results standardized against a double-priming, two-reaction protocol. Data, sourced from sgRNA-directed targeting of TRAC, EMX1, and VEGF3, are expressed as means with standard deviation. Statistical significance is calculated using paired two-tailed Student’s t tests, with adjusted p-values detailed for clarity.
