Fig. 2

Comparison of results from SMRT-OTS and Nano-OTS. a IGV alignments [38] showing the read distributions for SMRT-OTS (top) and Nano-OTS (bottom) in 20–50-kb windows spanning the ATXN10, MMP14, and NEK1 on-target sites. The Cas9 cleavage sites are clearly visible as sharp vertical lines where the alignments start or end. Usually, most of the reads are found either upstream or downstream of the Cas9 cleavage site and this imbalance is due to the orientation of the gRNA-Cas9 complex. Adapter ligation is less efficient on the 5′-side of the gRNA where the enzyme remains bound to the DNA after Cas9 cleavage [36]. b Examples of off-target alignment peaks for ATXN10 (left), MMP14 (middle), and NEK1 (right). At the top are sequence alignments between the gRNA sequence and the HEK293 genome at the off-target site. There are three single nucleotide mismatches at the ATXN10 and MMP14 off-target sites, and five single nucleotide mismatches at the NEK1 off-target site. All three off-target sites are visible in both in the SMRT-OTS and Nano-OTS alignment data. c Venn diagrams showing the overlap between gRNA predictions for SMRT-OTS and Nano-OTS for all three gRNAs combined (leftmost diagram) and each individual gRNA (the three other diagrams)