Figure 4

Method 

In order to obtain the 16S rRNA we had to extract and purify the soil DNA. We utilized the ZymoBIOMICS™ DNA MiniPrep kit from Zymo Research to extract the genomic DNA from our soil samples. We will use 750μL of the Lysis Solution and then vortex for 10 minutes. We then used a centrifuge and centrifuged the mixture in order to the supernatant that has our DNA. We filtered the Supernatant and then the flow through had our DNA. We continued this process until we were left with our DNA samples. 

We then measured the DNA Quality and Concentration by micro-pipetting our DNA samples into the Nanodrop pedestal. We also then sent our DNA to Rush University, and they performed sequencing on the 16S rRNA gene. 

Once we got our data results back, we used Nephele to obtain the full data results and get our taxonomic bar plot of the phyla and alpha biodiversity, and then we were able to get our richness, evenness, and Shannon diversity for our soil. 

Evidence

The data we are looking at is the average Shannon diversity index, richness, and evenness. We can see that after our data had been sequenced, we got different values than before. The average Shannon diversity index from our old rain garden is 4.605 and from our new is 4.6748. This shows there is only a 1.5% difference in the averages. The standard deviation was 0.0439 from the old and 0.00357 from our new.  For richness averages, we got 168.7 for the old rain garden and 148.3 for the new rain garden. This is a 12.87% difference in the averages for richness. The standard deviations for old and new were very similar. The old rain garden has a standard deviation of 4.92 and the new one of 4.77. Finally, we tested for evenness in the soil. The old rain garden had an average evenness of 0.921 and the new rain garden had an average of 0.912. This is only a 0.98% difference in the averages for evenness. The standard deviation for the old rain garden was 0.0042 and for the new was 0.0057. The P-value for all our tests came out to be less than 0.05 which rejects the null hypothesis and shows that our results are significantly different.

Conclusion

From the data in figure 4A, 4B, and 4C we see the Shannon diversity index, average richness, and average evenness for our new and old soil types. To determine if these samples have unique values, we can look at the p-values for each one. For the Shannon diversity index of the samples, the p-value is 0.00188. Since this value is below 0.05, this means that the Shannon diversity index of the samples is significantly different. Therefore, we can confidently conclude that the samples are unique, based on this p-value. For the average richness of the samples, our p-value was 5.80E-12. So, we can also conclude that they are significantly different. Our p-value for average evenness is 0.00574198, which is less than 0.05, proving that the samples are significantly different in evenness. We can conclude they are unique because these p-values are less than 0.05. Based on all these p-values, where they are all less than 0.05, we are confident in our data and are certain that the Shannon diversity index, average richness, average evenness of the old and new soil samples all have unique values and are all significantly different. 

Explanation

Derived from the conclusion in the previous paragraphs, we know that the rain gardens are significantly different and therefore unique as proven by the Shannon diversity index. A possible cause of this difference could be the “spatial patterns of microbial communities” within the soils (Naveed). A patchy distribution of species in the soils could lead to parts of the soil being more diverse than others. For example, if the sample from the old rain garden constituted more topsoil or soil with a larger than average amount of species, and the new garden sample was pulled from an area lacking diversity within the garden, then we would see results like this. Since the Shannon Index measures richness and evenness, the uneven distribution of topsoil and therefore species is very likely.