Barcode sequencing: a robust, platform-agnostic method for massively parallel cell-based screens.

Barcode sequencing (Bar-seq) is a high-throughput method originally developed for systematically identifying gene-drug interactions and genetic dependencies in yeast using pooled deletion-mutant libraries. This approach enables high-resolution profiling of large mutant libraries over time, across diverse experimental conditions, providing relative fitness values for each individual within the population. As the technology for enumerating barcodes has evolved, we have continued to incorporate improvements to the method. Here, we present an optimized Bar-seq workflow adaptable to multiple sequencing platforms, including instruments from Illumina, MGI, Element, and Oxford Nanopore. We highlight the advantages and limitations of each approach to aid in experimental design decisions. We introduce refinements in barcode amplification, sequencing strategies, and data analysis to enhance accuracy and scalability while making adoption as straightforward as possible.