Fig. 1

REMI-seq can be used to generate a comprehensive mutant collection in D. discoideum. A Schematic illustration of REMI mutageneisis. Insertion will take place if DpnII is used as the mutagenic enzyme together with BamHI digested linear plasmid. This will also work with NlaIII if the BamHI site is replaced with a SphI site. B In silico analysis suggests random genome-wide accessibility to REMI mutagenesis using a combination of restriction enzymes. Predicted DpnII and NlaIII sites are densely and randomly distributed across the genome. The density of each site was mapped onto 10kb fragments of the genome. Few regions are under- or overrepresented and thus represent putative cold or hotspots. C Schematic of the REMI-seq vector. pGWDI is a derivative of pLPBLP which encodes the blasticidin S deaminase selectable marker under the control of the actin15 promoter and actin8 terminator sequences. The sequence was modified (purple box) to contain sequences for REMI insertion. The pGWDI-C and pGWDI-G plasmids are identical with the exception that they have SphI and BamHI sites for integration. In addition, four different versions of each vector were generated with unique left and right 6bp sequence indices to allow increased multiplexing for insertion point analyses. D Schematic of insertion identification by REMI-seq. The pGWDI fragment is introduced in the D. discoideum genome by electroporation in the presence of DpnII or NlaIII. The termini of the insertion cassette contain recognition sites for MmeI and I-SceI which allows 20 bp fragments of gDNA at the insertion site to be generated. Red and blue boxes indicate vector-specific barcodes that allow multiplexing. Following the addition of adapter sequences, PCR amplification and Illumina sequencing, mapped reads can be aligned to the reference genome to identify insertion sites. Index sequences can be used to identify orientation/quantify the number of reads that map to each site provides quantitative information about the mutant abundance