Substrate enrichment level and concentration
Substrates used in SIP experiments are in almost all cases "fully" labelled, i.e. all positions are enriched with the labelled isotope to the highest level possible (> 97 at. %). This, of course, stems from the need to achieve high levels of labelling in the nucleic acids to detect labelled microbes. However, labelling of carbon only at specific positions has also been employed, for example to study microbial guilds that would attack the substrate at a specific position of interest, while excluding others, as in (REFS). The substrate concnetration can also affect the rate and strength of labelling, however presenting a sample with unrealistically high-concentrations can lead to undesired consequences such as drastic community changes or a rapid enrichment of a fast-growing sub-population with low substrate affinity. Therefore it is best to remain within the range (typcially on the higher end) of substrate concentrations that are expected to be found in the environment.
Number of fractions to collect and sequencing depth
Regardless of which method is used for analysing the data, success in a SIP experiment is determined by the ability to detect microbial phylotypes that are present in the denser fractions of a labelled gradient and are either absant or have lower abundance in the lighter fractions of the same gradient, or in the denser fraction of a control gradient. The detection limit in SIP experiments is itself not a fixed value but will depend on the sequencing depth, the number of fractions being collected from each gradient, and on which method is being used to analyse the data (see Section \ref{316470} for more details). Using advanced sequencing techonologies it is now easy to obtain thousands of sequences per fraction, however, this of course comes at a cost, which might not be necessary. It is therefore adviasable, if possible, to first obtain an estimate of the size of the microbial guild in question in relation to the total microbial population, using for example qPCR with primers targeting a funcitonal gene or fluorescent microscopy. The smaller the size of the target commuity, the harder it will be to detect its labelling above the detection limit. Naturally, this will almost inevitablly be and over estimation since only a part of the population will be active during the experiment and will eventually incorporate the substrate, but this will at least give a minimum threshold for the sequencing depth needed. The number of fractions collected can also affect the detection limit. While a higher number of fractions will most likely increase the sensitivity, it also entails higher sample processing efforts and costs. In addition, more fractions also mean less template per fraction and thus also an increased difficulty to amplify the target and a higher chance of contamination with foreign nucleic acids from the environment. Typically 12--20 fractions are collected, of which about 10--16 end up being analysed because the lightest and heavies fractions typically contain little to no nucleic acids.
Unlabelled controls
As in any lab experiment, appropriate label controls should be set up in parallel to minimise the detection of false-positives. Many of the older Particularly with the growing use of high-throughput sequencing and statistical models to detect labelled OTUs the need to include more no-label controls in the experiment in order to correctly detect labelled phylotypes has been growing. The exact number of no-label controls will depend on the exact statistical method used to analyse the data, but also on the type of SIP being performed since DNA-SIP is more prone to detecting false positives than RNA-SIP because of the effect of the G+C-content bias (see \ref{316470} for more details). Ideally, every labelled sample will have its parallel no-label control