Additional file 8: of UDiTaS™, a genome editing detection method for indels and genome rearrangements

Figure S7. UDiTaS characterization of plasmid standards without carrier DNA. To ensure that the carrier mouse genomic DNA was not influencing the UDiTaS reaction, additional sets of UDiTaS reactions were run with plasmids in the absence of any carrier DNA. a. CEP290 plasmids with the Wild Type, Large Deletion, and Large Insertion (PLA379, PLA367, and PLA370) and b. B2M-TRAC plasmids with the B2M, TRAC, and both balanced translocations (PLA377, PLA378, PLA365, and PLA366) were diluted as described in the methods. The DNA plasmids mixtures were process through UDiTaS and the analysis pipeline. Plotted is the expected frequency for a given structural variant vs. measured frequency for a structural variant (x = y is the grey line). Accuracy and linearity appear to be excellent for both loci with all four primers, with an LLOD of ~ 0.01%-0.1%. (PPTX 991 kb

Additional file 1: of UDiTaS™, a genome editing detection method for indels and genome rearrangements

Figure S1. Schematic of the bioinformatics pipeline for UDiTaS analysis. (PPTX 61 kb

Additional file 7: of UDiTaS™, a genome editing detection method for indels and genome rearrangements

Figure S6. UDiTaS characterization and comparison to AMP-Seq with plasmid standards. Plasmids containing the CEP290 structural variants a. or the TRAC-B2M balanced translocation b. and c. were synthesized and contain engineered unique SNPs in the insert to identify the plasmid after sequencing. The plasmids were diluted at various levels into mouse genomic DNA and processed through UDiTaS and AMP-Seq using primers for CEP290 a., B2M b. and TRAC c. The number of input plasmids versus the number of plasmids detected is plotted for both UDiTaS and AMP-Seq. Linear regression models and 95% confidence model predictions are displayed on the plots. The parameter β determines the linearity of the method, with values close to 1 indicating more linearity. We used ANOVA p-values to examine differences in β for UDiTaS and AMP-Seq. Below each plot, the table displays the total number of fastq reads sequenced in the reaction, the number of reads mapped to the wild-type amplicon (the most abundant one) and the final number of UMIs counted, for both UDiTaS and AMP-Seq. At all tested loci, UDiTaS shows greater linearity and number of UMIs detected when compared to AMP-Seq. (PPTX 12722 kb

Additional file 5: of UDiTaS™, a genome editing detection method for indels and genome rearrangements

Figure S4. Binomial power calculation applied to UDiTaS. A simulated binomial distribution, plotting editing frequency (e.g.: probability of success) vs. number of unique molecular identifiers (e.g.: trials) for a given number of expected observations (1, 2, or 3). Graphs on the left are 95% confidence and right 99% confidence. (PPTX 86 kb

Additional file 6: of UDiTaS™, a genome editing detection method for indels and genome rearrangements

Figure S5. Genome mapping rates for UDiTaS. Individual reads map to the expected genome site with high frequency indicating the robustness of the assay. Ten distinct samples for primer OLI6062 are plotted on the x-axis and the y-axis shows the percentage or reads mapping to the expected reference amplicon for each sample. (PPTX 3767 kb

Additional file 4: of UDiTaS™, a genome editing detection method for indels and genome rearrangements

Figure S3. UDiTaS reproducibility. Identical samples were run in UDiTaS using either SDS addition or Zymo column purification after tagmentation. Measured values for the various constructs are reproducible and highly correlated across a wide range of concentrations. (PPTX 3928 kb

Additional file 3: of UDiTaS™, a genome editing detection method for indels and genome rearrangements

Figure S2. Example editing events. Examples of various editing events in the U-2 OS bulk editing experiment shown in the Integrated Genome Viewer (IGV) [26, 27]. A schematic on top of each view depicts the observed editing event. Reads colored in red/blue were aligned to the top/bottom genomic reference DNA sequence. Note that small indels are observed in addition to the junctions formed from the larger structural changes. These indels likely arose due to repair pathway activity prior to rearrangement. a. 323 site small indels. b. 323-64 large desired 1.1 kb deletion junction. c. 323-64 large desired 1.1 kb inversion junction. d. 323 homologous junction. (PPTX 147 kb

Additional file 1: of Characterization of Staphylococcus aureus Cas9: a smaller Cas9 for all-in-one adeno-associated virus delivery and paired nickase applications

Supplementary figures and tables, which include plasmid maps, amino acid sequence alignments, indel data, and gRNA sequences. (DOCX 2159 kb