Figure 1: Illustration showing the hypothetical results of a 15N-SIP experiment analysed using different methods. A simplified community composed of only two phylotypes: phylotype 1 and phylotype 2 with genomic G+C contents of 51% and 67%, respectively. Each panel shows a simulated distribution of the two phylotypes in different SIP gradients. The x-axis shows the density of the fractions in the gradient while the y-axis shows the abundance of the two phylotypes in each fraction (e.g. obtained using qPCR). A. In a 14N-control DNA-SIP, phylotype 2 is centred around the denser parts of the gradient compared to phylotype 1 because of its higher G+C content. B. In a 15N-labelled DNA-SIP, only phylotype 1 incorporated the label and as a result migrated towards the denser fractions by about 0.016 g ml-1. However, this minor shift doesn't allow for visual separation from the unlabelled phylotype 2. Green arrows illustrate the binary comparison of the abundance (typically relative abundance) of each phylotype in its heavy fractions of a labelled gradient against its abundance in the heavy fractions of an unlabelled (control) gradient. Significantly higher relative abundance in the heavier fractions of a labelled gradient indicate labelling. Blue arrows illustrate the comparison done using qSIP where the mean shift of the buoyant density of each phylotype is calculated to determine its level of enrichment. C. In a secondary gradient using bis-benzimide both phylotypes migrate to the lower-density fractions but the low-G+C phylotype reduced its buoyant density more than the high-G+C phylotype and the two can be visually separated. D. In an RNA-SIP gradient, because G+C content has relatively little effect on buoyant density in the presence of formamide, the small mass addition from 15N labelling is visible. However, the statistical modelling using e.g. HR-SIP or qPCR will dramatically increase the detection power of the method (see Chapter 9 and 11 for further discussion.)