Prism v 8.2.1 for windows

After selection, AAV capsid sequences were recovered by PCR as described previously.²¹ (link) Twenty clones were sequenced after each round to track the progress of selection. The amino acid sequences of enriched AAV variants were aligned to known parental AAVs using Geneious Prime 2022.2.1 (https://www.geneious.com). This software was used to align sequences shown in Figure S1. A phylogenetic tree was drawn to scale, with branch lengths measured in the number of substitutions per site. Amino acid substitutions were also displayed using a heatmap generated from GraphPad Prism v.8.2.1 for Windows (GraphPad Software, Boston, MA). To visualize parental contribution in the capsids rAAV.KK01-05, the Xover tool was used (http://qpmf.rx.umaryland.edu/xover.html) for Figure 1C. Vectorization was then performed as described previously.²¹ (link) The novel capsid variants AAV.KK01-KK05 were then used to package one set of non-barcoded pAAV.CBA.GFP-expressing vectors, and another set of barcoded pAAV.CMV.GFP-BC-expressing vectors. AAV1, AAV6, and AAV9 were also included as control vectors. Vector packaging and titration was done as described previously.³⁵ (link) The capsids AAV.KK02, AAV.KK04, and AAV6 were also used to package pAAV.cTnT.GFP, which contains a cardiac specific promoter.

Student t test and Mann-Whitney U test were used to compare the differences in CWT and ADR between 2016 and 2019. One-way analysis of variance with Bonferroni post hoc test was used to compare different groups of endoscopists. Pearson correlation test was used to assess the relationship between the CWT and PDR. A two-tailed p value of <0.05 was considered significant. GraphPad Prism V.8.2.1 for Windows (GraphPad Software, La Jolla, California, USA) was used to conduct statistical analysis.