Fig. 2

Gridding and multiplexing of clonal mutants for sequencing. A Master grid contains 2304 wells. A grid containing six plates of mutants with the same barcode was generated. This was repeated for mutants with each GWDI barcode to make a stack of four grids. B Multiplexing of mutants for sequencing. Cells in each row (X), column (Y) and stack (Z) were pooled and gDNA prepared, to make a grid that is compressed into the X and Y coordinates. Unique combinations of X and Y barcoded sequencing adapters were ligated to each gDNA preparation before Illumina sequencing. The intersection of common mutants provides the well location for each mutant. The four mutants present in the compressed grid can be separated into stacking grids by their unique Z-barcode. C Intragenic insertions are biased towards the 5’ end of genes. The distance of each intragenic insertion from the start and stop codon was calculated and normalised to gene length as a percentage. 0% is the position of the start codon and 100% is the position of the stop codon. D The collection of individual D. discoideum REMI mutants. The REMI-seq mutant resource consists of 21,529 individual mutants. The majority of these mutations are in coding sequence or within putative promoter sequences and thus likely to result in gene disruptions. The REMI-seq resource consists of mutations in 5705 genes, resulting in a large increase in the number of mutants available. The catalogue of mutants can be found on the REMI-seq website (remi-seq.org) and individual mutants can be ordered from the Dictyostelium stock centre