# Chapter 2 Exprimental design(DoE)

Before you perform any metabolomic studies, a clean and meaningful experimental design is the best start. You need at least two groups: treated group and control group. Also you could treat this group infomation as the one primary variable or primary variables to be explored for certain research purposes.

The numbers of samples in each group should be carefully calculated. Supposing the metaoblites of certain biological process only have a few metabolites, the first goal of the exprimenal design is to find the differences of each metabolite in different group. For each metabolite, such comparision could be treated as one t-test. You need to perform a Power analysis to get the numbers. For example, we have two groups of samples with 10 samples in each group. Then we set the power at 0.9, which means 1 minus Type II error probability, the standard deviation at 1 and the significance level(Type 1 error probability) at 0.05. Then we get the meanful delta between the two groups should be higher than 1.53367 under this experiment design. Also we could set the delta to get the minimized numbers of the samples in each group. To get those data such as the standard deviation or delta for power analysis, you need to perform pre-experiments.

power.t.test(n=10,sd=1,sig.level = 0.05,power = 0.9)
##
##      Two-sample t test power calculation
##
##               n = 10
##           delta = 1.53367
##              sd = 1
##       sig.level = 0.05
##           power = 0.9
##     alternative = two.sided
##
## NOTE: n is number in *each* group
power.t.test(delta = 5,sd=1,sig.level = 0.05,power = 0.9)
##
##      Two-sample t test power calculation
##
##               n = 2.328877
##           delta = 5
##              sd = 1
##       sig.level = 0.05
##           power = 0.9
##     alternative = two.sided
##
## NOTE: n is number in *each* group

However, since sometimes we could not perform prelimintery experiment, we could directly compute the power based on false discovery rate control. If the power is lower than certain value, say 0.8, we just exclude this peak as significant features. Other study Blaise et al. (2016) show a method based on simulation to estimate the sample size. They usd BY correction to limit the influnces from correlationship. However, the nature of omics study make the power analysis hard to use one numbers and all the methods are trying to find a balance to represent more peaks with least samples(save money).

If there are other co-factors, a linear model or randomizing would be applied to eliminated their influences. You need to record the values of those co-factors for further data analysis. Common co-factors in metabolomic studies are age, gender, location, etc.

If you need data correction, some background or calibration samples are required. However, control samples could also be used for data correction in certain DoE.

Another important factors are instrumentals. High-resolution mass spectrum is always preferred. As shown in Lukas’s study Najdekr et al. (2016):

the most effective mass resolving powers for profiling analyses of metabolite rich biofluids on the Orbitrap Elite were around 60000–120000 fwhm to retrieve the highest amount of information. The region between 400–800 m/z was influenced the most by resolution.

However, elimination of peaks with high RSD% within group were always omited by most study. Based on pre-experiment, you could get a description of RSD% distribution and set cut-off to use stable peaks for further data analysis. To my knowledge, 50% is suitable considering the batch effects.

## 2.1 Software

• MetSizeR GUI Tool for Estimating Sample Sizes for Metabolomic Experiments.

### References

Blaise, Benjamin J., Gonçalo Correia, Adrienne Tin, J. Hunter Young, Anne-Claire Vergnaud, Matthew Lewis, Jake T. M. Pearce, et al. 2016. “Power Analysis and Sample Size Determination in Metabolic Phenotyping.” Anal. Chem. 88 (10): 5179–88. doi:10.1021/acs.analchem.6b00188.

Najdekr, Lukáš, David Friedecký, Ralf Tautenhahn, Tomáš Pluskal, Junhua Wang, Yingying Huang, and Tomáš Adam. 2016. “Influence of Mass Resolving Power in Orbital Ion-Trap Mass Spectrometry-Based Metabolomics.” Anal. Chem. 88 (23): 11429–35. doi:10.1021/acs.analchem.6b02319.