Highly replicated experiments studying complex genotypes using nested DNA barcodes

Many biological experiments involve studying the differences caused by genetic modifications, including genotypes composed of modifications at more than one locus. However, as the number and complexity of the genotypes increases, independently generating and tracking the necessary number of biological replicate samples becomes a major challenge. We developed a barcode-based method to track large numbers of independent replicates of combinatorial genotypes in a pooled format, enabling robust detection of subtle phenotypic differences. To construct a plasmid library of combinatorial genotypes, we utilized a nested serial cloning process to combine gene variants of interest that have associated DNA barcodes. The final plasmids each contain variants of multiple genes of interest, and a combined barcode that specifies the genotype of all the genes while also encoding a random sequence for tracking individual replicates. Sequencing of the pool of barcodes by next-generation sequencing allows the whole population to be studied in a single flask, enabling a high degree of replication even for complex genotypes. Using this approach, we tested the functionality of combinations of yeast, human, and null orthologs of the nucleotide excision repair factor I (NEF-1) complex and found that cells expressing all three yeast NEF-1 subunits had superior growth in DNA-damaging conditions. We also assessed the sensitivity of our method by simulating downsampling of barcodes across different degrees of phenotypic differentiation. Our results demonstrate the utility of NICR barcodes for high-throughput combinatorial genetic screens and provide a scalable framework for exploring complex genotype-phenotype relationships.