Sydney Gauss, Kaitlyn Ozcelebi, and Angelo Wong Determining Soil Ciliate Diversity: Optimal DNA Extraction Protocol for Metabarcoding Cili Cure Sydney Gauss, Kaitlyn Ozcelebi, and Angelo Wong Abstract Currently, the routes for examining species biodiversity in the soil have been deficient. This study is concerned with finding optimal protocols for determining the biodiversity of ciliates in the soil, which is an indicator of soil health. The purpose of this work is to develop a working protocol for DNA extraction of soil samples. With a sufficient protocol, the metabarcoding of purified DNA samples will be able to measure the diversity of environmental DNA (eDNA). The optimal protocol has been identified to be the modified Chelex DNA extraction. Other protocols such as MoBio’s PowerSoil Kit and ludox centrifugation have proven to be inadequate. The Chelex protocol uses chelating agents combined with centrifugation to obtain purified DNA. The V4 region, a variable region of the ribosomal 18S gene, was then amplified through PCR producing V4 PCR products 50% of the time. Eight out of 16 trials had V4 positive PCR products around 450 base pairs as shown by gel electrophoresis. This study is important to determine ciliate biodiversity for evaluating the well-being of food web interlinkages and biochemical cycles. While the modified Chelex protocol has shown to be effective half of the time, it provides the foundation for an ongoing investigation into further steps. Future goals include further developing of the protocol to more consistently obtain DNA for analysis. When developed, metabarcoding of the eDNA will allow the comparison of sequences to existing databases for species determination.

Determining Soil Ciliate Diversity: Optimal DNA Extraction Protocol for Metabarcoding Cili Cure Sydney Gauss, Kaitlyn Ozcelebi, and Angelo Wong Introduction Results Discussion Background Environmental biodiversity remains an important aspect in the state of soil health. The richness of species can determine environmental health due to their inherent linkages with food webs, energy processes, and nutrient cycles (Satyanarayana 2017). The scientific community lacks an effective route for the analysis of soil biodiversity. The standardization of a protocol has been impeded by the complexity of soil samples. Techniques such as bead beating and liquid nitrogen grinding seek to present an optimal design for DNA extraction and isolation. These techniques, however, lack the proper DNA purity from their samples and are expensive (Shuang 2013). The modified Chelex DNA extraction method uses chelating agents combined with centrifugation to obtain purified DNA. Chelex is a chelating agent that has high levels of affinity for metal ions preventing the nucleases of cells from destroying DNA components (Walsh 2013). Objective To develop a working protocol for DNA extraction of soil samples. To measure the diversity of environment DNA (eDNA) through the metabarcoding of purified DNA samples and gel electrophoresis. General Question How will the development of a sufficient protocol for DNA extraction and metabarcoding contribute to our knowledge of soil health? Figure 1: Gel electrophoresis of PCR products uses 1.8% agarose and ran DNA obtained from paramecium and Bermuda grass organisms. The DNA was extracted using the PowerSoil DNA extraction kit. Wells 2 and 5 contained the positive paramecium control and wells 3 and 6 contained the negative control. Wells 2-4 used primers Cox1 and Wells 5-7 used V4 primers. Well 1 contained DNAmark 100bp PLUS LADDER. BioRad Gel Doc EZ System and Image Lab software was used to image the gels. 1 2 3 5 4 6 7 8 Ludox Agarose Gel Electrophoresis Results 3,000 500 100 Size (bp) ng/5µl 40 Ludox centrifugation yielded negative results indicating that the strong ludox reagent destroyed the cell membrane of the organisms releasing their DNA prior to extraction. Due to ciliate fragility, a second trial with 2% glutaraldehyde was performed to fixate the cell membrane. Table 1 point to a significant absence of nucleic acid. Negative results are attributed to ethanol degrading the DNA (Zhao 2012). The PowerSoil protocol produced negative results due to the low nucleic acid concentration. Low DNA yields may also be accredited to residual ethanol contamination or the failure of the COX1 primer to bind to DNA (Hale 2015). The modified Chelex protocol has shown to be the most optimal protocol. V4 PCR products were produced 50% of the time with eight out of 16 trials showing V4 positive PCR products around 450 base pairs as shown by gel electrophoresis. This study is important to determine ciliate biodiversity for evaluating the well-being of food web interlinkages and biochemical cycles. While the modified Chelex protocol has shown to be effective half of the time, it provides the foundation for an ongoing investigation into further steps. The development of this working protocol enables researchers to obtain more purified DNA samples for more precise measurements of soil health. Future goals include further developing of the protocol to more consistently obtain DNA for analysis. When developed, metabarcoding of the eDNA will allow the comparison of sequences to existing databases for species determination. Nanodrop Results Nucleic Acid (ng/µl) A260/A280 A260/A230 Ludox -1.107 1.267 -0.046 PowerSoil 1.04 0.267 Chelex Trial 1 58.3 0.82 0.19 Chelex Trial 2 94.9 1.03 0.28 Table 1: The NanoDrop ND-1000 Specgtrophotometer was used to obtain the nucleic acid concentrations and absorbance levels of the trials shown in each figure. Figure 3: Gel electrophoresis of PCR products uses 1.8% agarose and ran DNA obtained from Bermuda soil in non-flooded plates. The DNA was extracted using the modified Chelex DNA extraction protocol. Wells 3 and 6 contained the positive control and wells 2 and 5 contained the negative control. Wells 2-7 used V4 primers. Well 1 contained DNAmark 100bp PLUS LADDER. BioRad Gel Doc EZ System and Image Lab software was used to image the gels. 1 2 3 5 4 6 7 8 Chelex Agarose Gel Electrophoresis Results 3,000 500 100 Size (bp) ng/5µl 40 Figure 2: Gel electrophoresis of PCR products uses 1.8% agarose and ran DNA obtained from paramecium and Bermuda grass organisms. The DNA was extracted using the PowerSoil DNA extraction kit. Wells 2 and 5 contained the positive paramecium control and wells 3 and 6 contained the negative control. Wells 2-4 used primers Cox1 and Wells 5-7 used V4 primers. Well 1 contained DNAmark 100bp PLUS LADDER. BioRad Gel Doc EZ System and Image Lab software was used to image the gels. 1 2 3 5 4 6 7 8 PowerSoil Agarose Gel Electrophoresis Results 3,000 500 100 Size (bp) ng/5µl 40 Methods Soil Collection: Bermuda grass sample PowerSoil: Soil DNA extraction kit; removal of PCR inhibitors PCR: Polymerase Chain Reaction amplified collected DNA V4 ribosomal primer and Cox1 mitochondrial primer Gel Electrophoresis: 1.8% Agarose Gel UV Gel analysis: visual test of collected DNA Non-Flooded Plate: Addition of water to petri dish to extract ciliates Chelex: Purfication of DNA through chelating agents Soil Juice: Washed soil solution made of soil and water mixture Ludox Centrifugation: Extraction of organic layer including ciliates References Hale, L., and David Crowley. (2015). DNA extraction methodology for biochar-amended sand and clay. Biology and Fertility of Soils, 51(6): 733-738, doi: 10.1007/s00374-015-1020-5 Morand, S., and Claire Lajaunie. Biodiversity and health: linking life, ecosystems, societies. ISTE Press, 2018. 9-12. Satyanarayana, Sadam D. V., et al. (2017). Optimization of high- Yielding protocol for DNA extraction from the forest rhizosphere microbes. 3 Biotech, 7(2), doi:10.1007/s13205-017-0737-2. Walsh, P. Sean, et al. (2013). Chelex 100 as a Medium for Simple Extraction of DNA for PCR-Based Typing form Forensic Material. BioTechniques, 54(3), doi: 10.2144/000114018 Zhao, F., et al. (2012). Application of the Ludox-QPS method for estimating ciliate diversity in soil and comparison with direct count and DNA fingerprinting. European Journal of Soil Biology, 49(1): 112- 118, doi: 10.1016/j.ejsobi.2011.11.002