Measurement error and resolution in quantitative stable isotope probing: implications for experimental design

Abstract

24Quantitative stable isotope probing (qSIP) estimates the degree of incorporation of an isotope 25 tracer into nucleic acids of metabolically active organisms and can be applied to microorganisms 26 growing in complex communities, such as the microbiomes of soil or water. As such, qSIP has 27 the potential to link microbial biodiversity and biogeochemistry. As with any technique 28 involving quantitative estimation, qSIP involves measurement error; a more complete 29 understanding of error, precision and statistical power will aid in the design of qSIP experiments 30 and interpretation of qSIP data. We used several existing qSIP datasets of microbial communities 31 found in soil and water to evaluate how variance in the estimate of isotope incorporation depends 32 on organism abundance and on the resolution of the density fractionation scheme. We also 33 assessed statistical power for replicated qSIP studies, and sensitivity and specificity for 34 unreplicated designs. We found that variance declines as taxon abundance increases. Increasing 35 the number of density fractions reduces variance, although the benefit of added fractions declines 36 as the number of fractions increases. Specifically, nine fractions appear to be a reasonable 37 tradeoff between cost and precision for most qSIP applications. Increasing replication improves 38 power and reduces the minimum detectable threshold for inferring isotope uptake to 5 atom%. 39Finally, we provide evidence for the importance of internal standards to calibrate the %GC to 40 mean weighted density regression per sample. These results should benefit those designing 41 future SIP experiments, and provide a reference for metagenomic SIP applications where 42 financial and computational limitations constrain experimental scope.43 44 45 46 3 Importance 47 48 One of the biggest challenges in microbial ecology is correlating the identity of microorganisms 49 with the roles they fulfill in natural environmental systems. Studies of microbes in pure culture 50 reveal much about genomic content and potential functions, but may not reflect an organism's 51 activity within its natural community. Culture-independent studies supply a community-wide 52 view of composition and function in the context of community interactions, but fail to link the 53 two. Quantitative stable isotope probing (qSIP) is a method that can link the identity and function 54 of specific microbes within a naturally occurring community. Here we explore how the 55 resolution of density-gradient fractionation affects the error and precision of qSIP results, how 56 they may be improved via additional replication, and cost-benefit balanced scenarios for SIP 57 experimental design. 58 59 65In SIP, a substrate labeled with a heavy isotope is added to an environmental sample. Following 66 an incubation period ranging from hours to weeks (depending on the substrate uptake rate) the 67 DNA (or RNA) of growing microorganisms that have consumed the isotope-enriched substrate 68 becomes more dense due to their incorp...

Stable isotope informed genome-resolved metagenomics uncovers potential trophic interactions in rhizosphere soil

“…The uncertainties of these estimated rates mirror the uncertainties in measuring the abundance of metagenome-assembled genomes in soil. The use of larger numbers of density fractions results in much reduced uncertainties in growth rate estimates (61). However, as expected, both phage and bacteria showed higher average growth rates than the slower-growing eukaryotes.…”

Active populations and growth of soil microorganisms are framed by mean annual precipitation in three California annual grasslands

“…Samples were subjected to a cesium chloride density gradient formed by physical density separation via ultracentrifugation as previously described with minor modifications 31,32 . For each sample, 5 μg of DNA in 150 μL 1xTE was mixed with 1.00 mL gradient buffer, and 4.60 mL CsCl stock (1.885 g mL -1 ) with a final average density of 1.730 g mL -1 .…”

“…We quantified excess atom fraction (EAF) 18 O of bacterial DNA following a modified version of the procedure described by Hungate et al using average DNA concentration to normalize the relative abundance of taxa within each density fraction 32,43,44 . We calculated median values and 95% confidence intervals for EAF 18 O by bootstrapping (n=1000) across experimental replicates.…”

Integrating Viral Metagenomics into an Ecological Framework

“…Heavy-water (H 2 18 O) SIP-metagenomics has recently gained a lot of attention because water is a universal substrate and incorporation of 18 O into newly synthesized DNA allows taxon-specific microbial growth and mortality rates to be calculated (95). Downsides to this method include the expensive cost of isotopically labeled substrate, the amount needed to ensure nucleic acid is sufficiently labeled, a high amount of nucleic acid required for input, and the cost and effort associated with sequencing multiple fractions as well as an unlabeled control sample to provide enough resolution for ecological inference (96).…”