Fig. 3: REMI-seq can be used to quantify the relative abundance of mutants.
From: The genetic architecture underlying prey-dependent performance in a microbial predator
a Schematic of amplified insert for REMI mutagenesis. The insert was PCR amplified from the pGWDI plasmid37. b The REMI-seq method for insertion point identification. The GWDI fragment containing a blasticidin resistance cassette (black) inserts into the D. discoideum genome at DpnII or NlaIII loci. The termini of the GWDI insertion fragment contain recognition sequences for the type II restriction enzyme MmeI (yellow) and the meganuclease I-SceI (blue). Digestion with MmeI and I-SceI results in the generation of two 47–48 bp fragments. These fragments can be enriched by PCR and gel extraction, thus allowing identification of 19/20 bp of genomic sequence at the site of insertion by sequencing. c REMI-seq exhibits high technical reproducibility. Three preparations of gDNA from a pool of ~13,000 mutants were sequenced. Normalised read counts for each mutant are highly correlated across each replicate (r = 0.92; r = 0.92; r = 0.91, p < 2.2 ×10−16 for all comparisons). d Technical reproducibility and dropout rate is dependent on read count. Mutants were divided into bins dependent on the average starting read count between all replicates of the starting library. The percentage of mutants that could not be detected (dropped out) in one or more library was calculated for each bin. e Schematic of the experiment to determine the quantitative dynamic range of REMI-seq. Cells of 32 defined mutants were distributed into four groups (A, B, C, D) of eight mutants each (Supplementary Data 1). 105, 106, 107, or 108 mutant cells from each group were mixed to form four different pools (P1–P4). f REMI-seq can be used to quantify mutant abundance over >100 fold range. Pools of mutants spiked at different frequencies (P1–P4) were subjected to REMI-seq. Violin plots show the number of cells (log10 transformed) was highly correlated with normalised read counts (r = 0.55, p = 2.439 ×10−11). All means are significantly different to each other (one-sided t-test, 105 vs 106: p = 0.002; 106 vs 107: p = 0.0002; 105 vs 106: p = 0.002; 107 vs 108: p = 0.0002). See Supplementary Data 1 for separate results of each group of mutants.
