Fig. 5

REMI-seq parallel phenotyping to identify mutants that affect axenic or bacterial growth. A Schematic of selections to identify mutants with growth advantages and disadvantages. A pool of ~20,000 mutants was plated in duplicate in HL5 medium for axenic growth or in association with K. aerogenes (Ka) bacteria. Cells were grown for 24 (axenic) or 100 generations (Ka) before a sample was harvested for sequencing. The remaining cells were diluted and grown-up for a further 24 (axenic) or 100 (Ka) generations before a second sample was taken for sequencing. Axenic cells were diluted and grown-up for a further 24 generations before sequencing. B Identification of mutants with growth advantages and disadvantages. The abundance (read count) at each round of each mutant was compared to the start pool. Mutants were first divided into bins based on their read count in the start pool in order to identify mutants that deviated in abundance (Z score) significantly from other mutants with similar read counts. Mutants with a mean Z score of >1.5 and with a minimum of 100 reads in each replicate at the end of the selection were considered to have increased in abundance (red points). Mutants with a mean Z score of < -1.0 (blue points) were considered to have decreased. In bin <100, the variation due to technical dropouts resulted in a high false discovery rate, and mutants that decreased were not considered. Replicas are highly correlated. Results are shown at the end point of the selection (after 200 generations of Ka growth and 72 generations of axenic growth). Data from other rounds showed similar patterns (see Additional files 8 and 9). C Mutant behaviour is consistent across rounds of selection. Mutants were identified with a significant advantage or disadvantage at each stage of the selection. The level of enrichment or depletion (deviation from expected or Z score) was plotted for each mutant at all rounds