This report illustrates a pipeline processing total internal reflection fluorescence (TIRF) microscope images combined with information from next-generation sequencing (NGS) technology. The TIRF movies recorded the single-molecule fluorescence resonance energy transfer (smFRET) occurring within every single molecule, while the NGS gave the sequence tag of those molecules. By doing coordinates registration, information from both of these technologies was combined with scaling the biophysical kinetic monitoring into the single molecule level. This pipeline also attempts to use machine learning for library-scale smFRET signal sequences. This pipeline has achieved basic automation, and in this report the gap between the fully automated system is also discussed as well as possible directions for future improvement.