16S Sequencing of CrystalSea No Template Control 336 Hours A 4C Sterile Water
This time course experiment was designed to monitor a controlled system for eDNA degradation under room temperature and refrigerated 4C conditions. Samples were measured in sterile and non-sterile water to determine the role of micro-organisms in the rate of degradation. Extracted DNA from the water was measured using qPCR for mitochondrial markers and 16S markers. Samples were also plated on Marine Agar plates to observe microbial activity and amplified using V1-V2 primers for 16S sequencing. Artificial seawater was created (CrystalSea) and thoroughly mixed before being poured into 500 mL aliquots in one liter Nalgene bottles for cell spike-in experiments. Sterile samples were autoclaved for 60 min. For the temporal degradation testing, 500mL aliquots in triplicates were used for each time point and water condition. Room temperature replicates were placed on the laboratory bench and 4C samples were placed in the refrigerator for >24 hours before addition of cells. For these experiments, HAM001 cell lines were counted and concentrations were normalized to 105 cells/L water and added to the seawater samples. No template sampling (NTC) controls in triplicate included the same seawater with no cell spike-ins for both timepoint 0 and timepoint 2 weeks (336 hours). Three bottles were filled for each of the timepoints with cells: 0, 1, 2, 3, 4, 5, 6, 7, 8, 24, 48, 72, 168 and 336 hours after spike-in. After sample spike-ins occurred, samples were then immediately filtered (except in the case of temporal samples) using 0.45M Sterivexfilters (Millipore) and a diaphragm pump connected to inlet and outlet hoses that were cleaned with 70% ethanol between each individual samples. After filtration, filters were kept at -80C until DNA extraction. eDNA was extracted from the filters using the DNeasy Blood and Tissue Kit (Qiagen). Modifications were made to the standard protocol as follows: 720L of Buffer ATL was added to the filter cartridge, along with 80L of Proteinase K (20mg/mL, SigmaAldrich) before being incubated at 56C for 24 hours using end-over-end rotation. Equal parts 200 proof MBG ethanol and Buffer AL were added to samples after removal from the filter cartridge, then processed according to the manufacturer protocol. Extracted DNA samples were quantified using a Qubit dsDNA HS assay (ThermoFisher Scientific). Samples were amplified for absolute mitochondrial copy number counts using the human mithcondrial DNA (mtDNA) monitoring primer set (Takara Bio). This qPCR set contains two mitochondrial marker primers (ND1 and ND5) and two nuclear marker primers (SLC02B1 and SERPINA1). Only the mitochondrial markers were used for this set. The PCR reaction consisted of 10L of PowerUp SYBR Green Master Mix (ThermoFisher), 7L nuclease free water (NFW), 1L of each appropriate primer mix at 10M, and 2L of template DNA. Each triplicate sample was amplified for the two mitochondrial markers using the following thermal profile on a QuantStudio 6 Pro: 95C for 30s, followed by 40 cycles of 95C for 5s and 60C for 30s, followed by a melt curve analysis. Threshold cycle (Ct) information was recovered from the run analysis and used to calculate the degradation of samples over time. Extracted DNA from all replicates from NTC 0, T0, T8, T168, T336, and NTC 336 for all conditions were amplified using the V1-V2 primer sets (F: TCGTCGGCAGCGTCAGATGTGTATAAGAGACAGAGAGTTTGATCCTGGCTCAG, R: GTCTCGTGGGCTCGGAGATGTGTATAAGAGACAGGCTGCCTCCCGTAGGAGT) and regions and indexed using the Nextera XT library preparation kit and sequenced on a 600 cycle V1 kit for the Nextseq 2000. Samples were loaded at 650 pM concentration and PhiX was spiked in at 50% to balance amplicon reads.