Extended Data Fig. 4: Cas9-dependent off-target sites are verified by an optimized targeted amplicon sequencing strategy.
From: Detect-seq reveals out-of-protospacer editing and target-strand editing by cytosine base editors
a, Schematic workflow of the improved targeted amplicon sequencing procedure. Extended unique molecular identifiers (UMIs) are introduced to mark each amplicon during the first round of PCR amplification. b, The bioinformatic strategy to remove PCR duplicates and errors generated during the second round of PCR amplification according to left and right UMIs. c, d, Matched targeted amplicon sequencing results for Fig. 1e (c) and in Supplementary Fig. 4b (d). e, Detect-seq and the matched targeted high-throughput sequencing results were given for a representative putative sgRNA-binding site for the constructs in Fig. 1c. The pRBS is shadowed. Green and red blocks in the IGV views respectively indicate C-to-T mutations on the non-target strand and target strand; the red and green inverted triangles respectively indicate genuine C-to-T edits on the forward and reverse strand according to the results of targeted amplicon sequencing.
