Abstract
DNA Stable isotope probing (DNA-SIP) is a powerful method that links identity to function within microbial communities. The combination of DNA-SIP with multiplexed high throughput DNA sequencing enables simultaneous mapping of in situ assimilation dynamics for thousands of microbial taxonomic units. Hence, high throughput sequencing enabled SIP has enormous potential to reveal patterns of carbon and nitrogen exchange within microbial food webs. There are several different methods for analyzing DNA-SIP data and despite the power of SIP experiments, it remains difficult to comprehensively evaluate method accuracy across a wide range of experimental parameters. We have developed a toolset (SIPSim) that simulates DNA-SIP data, and we use this toolset to systematically evaluate different methods for analyzing DNA-SIP data. Specifically, we employ SIPSim to evaluate the effects that key experimental parameters (e.g., level of isotopic enrichment, number of labeled taxa, relative abundance of labeled taxa, community richness, community evenness, and beta-diversity) have on the specificity, sensitivity, and balanced accuracy (defined as the product of specificity and sensitivity) of DNA-SIP analyses. Furthermore, SIPSim can predict analytical accuracy and power as a function of experimental design and community characteristics, and thus should be of great use in the design and interpretation of DNA-SIP experiments.
Highlights
Stable isotope probing of nucleic acids (DNA-SIP and RNA-SIP) is a powerful culture-independent method for linking microbial metabolic functioning to taxonomic identity (Radajewski et al, 2003)
Isopycnic centrifugation is distinguished from other centrifugation methods in that centrifugation is carried out long enough to both generate a density gradient and allow all macromolecules of interest reach sedimentation equilibrium, which is the point at which sedimentation rates equal rates of diffusion (Hearst and Schmid, 1973; Birnie and Rickwood, 1978)
The genomes are fragmented as would occur during DNA extraction, isotopic labeling is applied to some number of genomes as specified by the user, the buoyant density (BD) distributions are determined for each DNA fragment and fragment collections are binned into gradient fractions, fragments are sampled from each fraction as would occur during amplification and DNA sequencing of SSU rRNA genes, and the relative abundance is calculated for each OTU (Figure 1, see section Methods)
Summary
Stable isotope probing of nucleic acids (DNA-SIP and RNA-SIP) is a powerful culture-independent method for linking microbial metabolic functioning to taxonomic identity (Radajewski et al, 2003). DNA-SIP has been used in a multitude of environments to identify microbial assimilation of various 13C- and 15N-labeled substrates into DNA (Uhlík et al, 2009). DNA-SIP identifies microbes that assimilate these isotopes into their DNA (“incorporators”) by exploiting the increased buoyant density (BD) of isotopically labeled (“heavy”) DNA relative to unlabeled (“light”) DNA. Fully 13C- and 15N-labeled DNA will increase in BD by 0.036 and 0.016 g ml−1, respectively (Birnie and Rickwood, 1978). Isopycnic centrifugation could be used to completely separate labeled and unlabeled DNA fragments based solely on this difference in BD.
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