scholarly journals Measurement Error and Resolution in Quantitative Stable Isotope Probing: Implications for Experimental Design

mSystems ◽  
2020 ◽  
Vol 5 (4) ◽  
Author(s):  
Ella T. Sieradzki ◽  
Benjamin J. Koch ◽  
Alex Greenlon ◽  
Rohan Sachdeva ◽  
Rex R. Malmstrom ◽  
...  
Keyword(s):  
Measurement Error ◽  
Experimental Design ◽  
Stable Isotope ◽  
Statistical Power ◽  
Quantitative Estimation ◽  
Cost Benefit ◽  
Data Interpretation ◽  
Stable Isotope Probing ◽  
Isotope Enrichment ◽  
Community Interactions

ABSTRACT Quantitative stable isotope probing (qSIP) estimates isotope tracer incorporation into DNA of individual microbes and can link microbial biodiversity and biogeochemistry in complex communities. As with any quantitative estimation technique, qSIP involves measurement error, and a fuller understanding of error, precision, and statistical power benefits qSIP experimental design and data interpretation. We used several qSIP data sets—from soil and seawater microbiomes—to evaluate how variance in isotope incorporation estimates depends on organism abundance and resolution of the density fractionation scheme. We assessed statistical power for replicated qSIP studies, plus sensitivity and specificity for unreplicated designs. As a taxon’s abundance increases, the variance of its weighted mean density declines. Nine fractions appear to be a reasonable trade-off between cost and precision for most qSIP applications. Increasing the number of density fractions beyond that reduces variance, although the magnitude of this benefit declines with additional fractions. Our analysis suggests that, if a taxon has an isotope enrichment of 10 atom% excess, there is a 60% chance that this will be detected as significantly different from zero (with alpha 0.1). With five replicates, isotope enrichment of 5 atom% could be detected with power (0.6) and alpha (0.1). Finally, we illustrate the importance of internal standards, which can help to calibrate per sample conversions of %GC to mean weighted density. These results should benefit researchers designing future SIP experiments and provide a useful reference for metagenomic SIP applications where both financial and computational limitations constrain experimental scope. IMPORTANCE One of the biggest challenges in microbial ecology is correlating the identity of microorganisms with the roles they fulfill in natural environmental systems. Studies of microbes in pure culture reveal much about their genomic content and potential functions but may not reflect an organism’s activity within its natural community. Culture-independent studies supply a community-wide view of composition and function in the context of community interactions but often fail to link the two. Quantitative stable isotope probing (qSIP) is a method that can link the identity and functional activity of specific microbes within a naturally occurring community. Here, we explore how the resolution of density gradient fractionation affects the error and precision of qSIP results, how they may be improved via additional experimental replication, and discuss cost-benefit balanced scenarios for SIP experimental design.

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

2020 ◽  
Author(s):  
Ella T. Sieradzki ◽  
Benjamin J. Koch ◽  
Alex Greenlon ◽  
Rohan Sachdeva ◽  
Rex R. Malmstrom ◽  
...  
Keyword(s):  
Measurement Error ◽  
Experimental Design ◽  
Stable Isotope ◽  
Statistical Power ◽  
Quantitative Estimation ◽  
Cost Benefit ◽  
Stable Isotope Probing ◽  
Community Interactions ◽  
Density Fractions ◽  
And Function

AbstractQuantitative stable isotope probing (qSIP) estimates the degree of incorporation of an isotope tracer into nucleic acids of metabolically active organisms and can be applied to microorganisms growing in complex communities, such as the microbiomes of soil or water. As such, qSIP has the potential to link microbial biodiversity and biogeochemistry. As with any technique involving quantitative estimation, qSIP involves measurement error; a more complete understanding of error, precision and statistical power will aid in the design of qSIP experiments and interpretation of qSIP data. We used several existing qSIP datasets of microbial communities found in soil and water to evaluate how variance in the estimate of isotope incorporation depends on organism abundance and on the resolution of the density fractionation scheme. We also assessed statistical power for replicated qSIP studies, and sensitivity and specificity for unreplicated designs. We found that variance declines as taxon abundance increases. Increasing the number of density fractions reduces variance, although the benefit of added fractions declines as the number of fractions increases. Specifically, nine fractions appear to be a reasonable tradeoff between cost and precision for most qSIP applications. Increasing replication improves power and reduces the minimum detectable threshold for inferring isotope uptake to 5 atom%. Finally, we provide evidence for the importance of internal standards to calibrate the %GC to mean weighted density regression per sample. These results should benefit those designing future SIP experiments, and provide a reference for metagenomic SIP applications where financial and computational limitations constrain experimental scope.ImportanceOne of the biggest challenges in microbial ecology is correlating the identity of microorganisms with the roles they fulfill in natural environmental systems. Studies of microbes in pure culture reveal much about genomic content and potential functions, but may not reflect an organism’s activity within its natural community. Culture-independent studies supply a community-wide view of composition and function in the context of community interactions, but fail to link the two. Quantitative stable isotope probing (qSIP) is a method that can link the identity and function of specific microbes within a naturally occurring community. Here we explore how the resolution of density-gradient fractionation affects the error and precision of qSIP results, how they may be improved via additional replication, and cost-benefit balanced scenarios for SIP experimental design.

scholarly journals Stable Isotope Probing with 15N Achieved by Disentangling the Effects of Genome G+C Content and Isotope Enrichment on DNA Density

2007 ◽  
Vol 73 (10) ◽  
pp. 3189-3195 ◽  
Author(s):  
Daniel H. Buckley ◽  
Varisa Huangyutitham ◽  
Shi-Fang Hsu ◽  
Tyrrell A. Nelson
Keyword(s):  
Stable Isotope ◽  
Natural Variation ◽  
Buoyant Density ◽  
Stable Isotope Probing ◽  
Density Gradients ◽  
Isotope Enrichment ◽  
Labeled Compounds ◽  
Contrast Variation ◽  
Functional Capabilities ◽  
Dna Density

ABSTRACT Stable isotope probing (SIP) of nucleic acids is a powerful tool that can identify the functional capabilities of noncultivated microorganisms as they occur in microbial communities. While it has been suggested previously that nucleic acid SIP can be performed with 15N, nearly all applications of this technique to date have used 13C. Successful application of SIP using 15N-DNA (15N-DNA-SIP) has been limited, because the maximum shift in buoyant density that can be achieved in CsCl gradients is approximately 0.016 g ml−1 for 15N-labeled DNA, relative to 0.036 g ml−1 for 13C-labeled DNA. In contrast, variation in genome G+C content between microorganisms can result in DNA samples that vary in buoyant density by as much as 0.05 g ml−1. Thus, natural variation in genome G+C content in complex communities prevents the effective separation of 15N-labeled DNA from unlabeled DNA. We describe a method which disentangles the effects of isotope incorporation and genome G+C content on DNA buoyant density and makes it possible to isolate 15N-labeled DNA from heterogeneous mixtures of DNA. This method relies on recovery of “heavy” DNA from primary CsCl density gradients followed by purification of 15N-labeled DNA from unlabeled high-G+C-content DNA in secondary CsCl density gradients containing bis-benzimide. This technique, by providing a means to enhance separation of isotopically labeled DNA from unlabeled DNA, makes it possible to use 15N-labeled compounds effectively in DNA-SIP experiments and also will be effective for removing unlabeled DNA from isotopically labeled DNA in 13C-DNA-SIP applications.

scholarly journals Quantitative Microbial Ecology through Stable Isotope Probing

2015 ◽  
Vol 81 (21) ◽  
pp. 7570-7581 ◽  
Author(s):  
Bruce A. Hungate ◽  
Rebecca L. Mau ◽  
Egbert Schwartz ◽  
J. Gregory Caporaso ◽  
Paul Dijkstra ◽  
...  
Keyword(s):  
Stable Isotope ◽  
Isotope Labeling ◽  
Stable Isotope Probing ◽  
Taxonomic Resolution ◽  
Isotope Enrichment ◽  
Density Fractions ◽  
Isotope Tracers ◽  
Isotope Tracer ◽  
Quantitative Measurements ◽  
Isopycnic Centrifugation

ABSTRACTBacteria grow and transform elements at different rates, and as yet, quantifying this variation in the environment is difficult. Determining isotope enrichment with fine taxonomic resolution after exposure to isotope tracers could help, but there are few suitable techniques. We propose a modification tostableisotopeprobing (SIP) that enables the isotopic composition of DNA from individual bacterial taxa after exposure to isotope tracers to be determined. In our modification, after isopycnic centrifugation, DNA is collected in multiple density fractions, and each fraction is sequenced separately. Taxon-specific density curves are produced for labeled and nonlabeled treatments, from which the shift in density for each individual taxon in response to isotope labeling is calculated. Expressing each taxon's density shift relative to that taxon's density measured without isotope enrichment accounts for the influence of nucleic acid composition on density and isolates the influence of isotope tracer assimilation. The shift in density translates quantitatively to isotopic enrichment. Because this revision to SIP allows quantitative measurements of isotope enrichment, we propose to call it quantitative stable isotope probing (qSIP). We demonstrated qSIP using soil incubations, in which soil bacteria exhibited strong taxonomic variations in18O and13C composition after exposure to [18O]water or [13C]glucose. The addition of glucose increased the assimilation of18O into DNA from [18O]water. However, the increase in18O assimilation was greater than expected based on utilization of glucose-derived carbon alone, because the addition of glucose indirectly stimulated bacteria to utilize other substrates for growth. This example illustrates the benefit of a quantitative approach to stable isotope probing.

scholarly journals Quantitative microbial ecology through stable isotope probing

2015 ◽  
Author(s):  
Bruce A Hungate ◽  
Rebecca L Mau ◽  
Egbert Schwartz ◽  
J Gregory Caporaso ◽  
Paul Dijkstra ◽  
...  
Keyword(s):  
Stable Isotope ◽  
Isotope Labeling ◽  
Stable Isotope Probing ◽  
Taxonomic Resolution ◽  
Isotope Enrichment ◽  
Glucose Addition ◽  
Density Fractions ◽  
Isotope Tracers ◽  
Isotope Tracer ◽  
Quantitative Measurements

Bacteria grow and transform elements at different rates, yet quantifying this variation in the environment is difficult. Determining isotope enrichment with fine taxonomic resolution after exposure to isotope tracers could help, but there are few suitable techniques. We propose a modification to Stable Isotope Probing (SIP) that enables determining the isotopic composition of DNA from individual bacterial taxa after exposure to isotope tracers. In our modification, after isopycnic centrifugation, DNA is collected in multiple density fractions, and each fraction is sequenced separately. Taxon specific density curves are produced for labeled and non-labeled treatments, from which the shift in density for each individual taxon in response to isotope labeling is calculated. Expressing each taxon’s density shift relative to that taxon’s density measured without isotope enrichment accounts for the influence of nucleic acid composition on density and isolates the influence of isotope tracer assimilation. The shift in density translates quantitatively to isotopic enrichment. Because this revision to SIP allows quantitative measurements of isotope enrichment, we propose to call it quantitative Stable Isotope Probing (qSIP). We demonstrate qSIP using soil incubations, in which soil bacteria exhibited strong taxonomic variation in 18O and 13C composition after exposure to 18O-H2O or 13C-glucose. Addition of glucose increased assimilation of 18O into DNA from 18O-H2O. However, the increase in 18O assimilation was greater than expected based on utilization of glucose-derived carbon alone, because glucose addition indirectly stimulated bacteria to utilize other substrates for growth. This example illustrates the benefit of a quantitative approach to stable isotope probing.

scholarly journals Quantitative microbial ecology through stable isotope probing

2015 ◽  
Author(s):  
Bruce A Hungate ◽  
Rebecca L Mau ◽  
Egbert Schwartz ◽  
J Gregory Caporaso ◽  
Paul Dijkstra ◽  
...  
Keyword(s):  
Stable Isotope ◽  
Isotope Labeling ◽  
Stable Isotope Probing ◽  
Taxonomic Resolution ◽  
Isotope Enrichment ◽  
Glucose Addition ◽  
Density Fractions ◽  
Isotope Tracers ◽  
Isotope Tracer ◽  
Quantitative Measurements

Bacteria grow and transform elements at different rates, yet quantifying this variation in the environment is difficult. Determining isotope enrichment with fine taxonomic resolution after exposure to isotope tracers could help, but there are few suitable techniques. We propose a modification to Stable Isotope Probing (SIP) that enables determining the isotopic composition of DNA from individual bacterial taxa after exposure to isotope tracers. In our modification, after isopycnic centrifugation, DNA is collected in multiple density fractions, and each fraction is sequenced separately. Taxon specific density curves are produced for labeled and non-labeled treatments, from which the shift in density for each individual taxon in response to isotope labeling is calculated. Expressing each taxon’s density shift relative to that taxon’s density measured without isotope enrichment accounts for the influence of nucleic acid composition on density and isolates the influence of isotope tracer assimilation. The shift in density translates quantitatively to isotopic enrichment. Because this revision to SIP allows quantitative measurements of isotope enrichment, we propose to call it quantitative Stable Isotope Probing (qSIP). We demonstrate qSIP using soil incubations, in which soil bacteria exhibited strong taxonomic variation in 18O and 13C composition after exposure to 18O-H2O or 13C-glucose. Addition of glucose increased assimilation of 18O into DNA from 18O-H2O. However, the increase in 18O assimilation was greater than expected based on utilization of glucose-derived carbon alone, because glucose addition indirectly stimulated bacteria to utilize other substrates for growth. This example illustrates the benefit of a quantitative approach to stable isotope probing.

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