Sequencing of FISH-Barcoded Plasmid Libraries

Optical Pooled Screening (OPS) enables precise measurement of pooled genetic libraries in single, highly multiplexed microscopy experiments, linking image-based phenotypes to genotypes at genome-scale. However, no method has combined OPS with multi-generational timelapse imaging, which is especially important for studying single-cell dynamics and physiology in microbes. Here we develop MARLIN, a technique for multigenerational timelapse OPS in a microfluidic device and leveraged it map live-cell imaging data to genetic perturbations in a ~30k member mismatch-CRISPRi library targeting all essential genes in E. coli. The resulting dataset comprises nearly a hundred million cell cycles, represents an average of 33 different knockdowns per gene, and provides quantitative measures of morphology and growth for each essential gene with respect to dose. The sequencing data associated with this study correspond to amplicons of various FISH barcode-labeled plasmid libraries, which were sequenced by an Oxford Nanopore. These were used to build a lookup table between the FISH barcode and the underlying genetic variants of interest.