REMI-seq generation of a genome-wide mutant resource for dictyostelium functional genomics

We are combining Restriction Enzyme Mediated Integration (REMI) mutagenesis with NGS technology to create a genome-wide set of gene knockout mutants. Using this process, we aim to create a near total collection of Dictyostelium mutants as a new resource for the Dictyostelium community. The resource will comprise individual, sets and large pools of mutants, with the loci of REMI mutations searchable via dictyBase. To date, we have generated and stored more than 11,000 insertion mutants by REMI. Insertion sites are identified by sequencing the insert/genomic DNA junction. The junctions are captured using MmeI, a type IIC restriction enzyme; indexed with sequencing adapters and amplified by PCR. Following size selection, the target DNA is sequenced using an Illumina MiSeq. A Bioinformatic pipeline has been designed to interpret the data and catalogue the mutants. So far, the REMI insertion sites for ~1,000 mutants have been identified, including 280 mutants with ‘in-gene’ insertions that are not currently catalogued in the dictyBase stock centre. 84% of the stocks contain a single mutant with a single insertion. 63% have in-gene insertions and 4% contain insertions attributed to hotspots. This new resource will produce a step change in Dictyostelium genetics. The principle benefits will be the on-line availability of independent and multi-allelic mutants for nearly all Dictyostelium genes, the ease of whole genome phenotypic screens and lastly the capacity to conduct complex phenotyping of protein families.