Figure 2. iRep is an accurate measure of in situ replication rates.

(a) iRep, bPTR, and kPTR measurements made for cultured Lactobacillus gasseri⁸ were compared (r = Pearson’s r value), showing strong agreement between all methods. (b) Colony forming unit (CFU) counts were available for a subset of these samples⁸, and used to calculate growth rates (n = 2). All methods were highly correlated with CFU-derived rates after first accounting for the delay between start of genome replication and observable change in population size (as noted previously⁸). Replication rates from CFU data were adjusted by variable amounts before calculating correlations with sequencing-based rates (best correlation shown; d = time adjustment). CFU data are plotted with a −90 minute offset. (c) Using the L. gasseri data, minimum coverage requirements were determined for each method by first measuring the replication rate at 25× coverage, and then comparing to values calculated after simulating lower coverage. This shows that ≥5× coverage is required. (d) The minimum required genome fraction for iRep was determined by conducting 100 random fragmentations and subsets of the L. gasseri genome. Sequencing was subset to 5× coverage before calculating iRep to show the combined affect of low coverage and missing genomic information. With ≥75% of a genome sequence, most iRep measurements are accurate ±0.15. (e) iRep and bPTR measurements were calculated using five genome sequences assembled from premature infant metagenomes, showing that these methods are in agreement in the context of microbiome sequencing data.