This work contains the experimental data and associated analysis that are described in the research publication entitled "Ultra-specific and Amplification-free Quantification of Mutant DNA by Single-molecule Kinetic Fingerprinting". This work contains multiple zip files, each of which represents one of the principal experiment groups presented in the publication. Each experiment group contains movie and analysis files corresponding to various experimental conditions related to that experiment group.
Traces from single-molecule fluorescence microscopy (SMFM) experiments exhibit photophysical artifacts that typically necessitate human expert screening, which is time-consuming and introduces potential for user-dependent expectation bias. Here, we have used deep learning to develop a rapid, automatic SMFM trace selector, termed AutoSiM, that improves the sensitivity and specificity of an assay for a DNA point mutation based on single-molecule recognition through equilibrium Poisson sampling (SiMREPS). The improved performance of AutoSiM is based on accepting both more true positives and fewer false positives than the conventional approach of hidden Markov modeling (HMM) followed by thresholding. As a second application, the selector was used for automated screening of single-molecule Förster resonance energy transfer (smFRET) data to identify high-quality traces for further analysis, and achieves ~90% concordance with manual selection while requiring less processing time. AutoSiM can be adapted readily to novel datasets, requiring only modest Transfer Learning.
Li, J., Zhang, L., Johnson-Buck, A., & Walter, N. G. (2020). Automatic classification and segmentation of single-molecule fluorescence time traces with deep learning. Nature Communications, 11(1), 5833. https://doi.org/10.1038/s41467-020-19673-1 and Hayward, S., Lund, P., Kang, Q., Johnson-Buck, A., Tewari, M., Walter, N. (2018). Single-molecule microscopy image data and analysis files for "Ultra-specific and Amplification-free Quantification of Mutant DNA by Single-molecule Kinetic Fingerprinting" [Data set]. University of Michigan - Deep Blue. https://doi.org/10.7302/Z2CZ35DF
