A multiplexed, three-dimensional pooling and next generation sequencing strategy for construction of a barcoded Schizosaccharomyces pombe transposon insertion library

We developed a novel three-dimensional pooling strategy and multiplexed high-throughput analysis pipeline to sequence the transposon insertion sites and DNA barcodes from thousands of samples simultaneously. The library contains 4,095 mutants with insertions in the gene and UTRs of non-essential genes, essential genes, and non-coding RNA genes. Selected mutants were tested for growth on medium containing a non-fermentable carbon source or topoisomerase I inhibitor CPT, which revealed that, while some insertion mutants have the same phenotypes as the cognate deletion mutants, others have novel phenotypes. This library represents an important resource for the international S. pombe community and a valuable approach for construction and analysis of insertion mutant libraries in a wide variety of model systems.Methods: 3D pooling and multiplexed deep sequencing to map transposon integrations and DNA barcodes. 3D pooling: 24 plates (2,256 mutants) were stacked as 2 plates per layer, a total of 12 layers. Cells were collected in the format of a Row Pool (144 strains/pool), a Column Pool (196 strains/pool) and a Layer Pool (192 strains/pool). A total of 40 pools of cells were collected, including 16 Column Pools, 12 Row Pools and 12 Layer Pools. Amplification of Hermes insertion sites and barcodes: Genomic DNA from a row, column or layer pool was fragmented by restriction enzyme digestion and amplified to capture Hermes transposon and barcode-specific sequences. To index the pools, primers were synthesized with 8-mer tags, and the tag sequences were removed prior to analysis. Analysis pipeline: After sequencing, flanking genomic sequences at both ends of the transposon were aligned to the S. pombe reference genome to map the insertion and the sequences common to a row, column and layer pool identified a mutant in a single well of a single 96-well plate.