The validation run took place March-April 2015, in the Agua Hedionda Lagoon, Carlsbad, CA. Reason to choose this location was the ease to deploy and collect samples attached to a dock. Our goals were to test the sterilization method of the plastics (ethanol washes and UV irradiation), control for possible contamination due to manipulation when setting up the cages and collection, as well as during DNA extraction. We also wanted to pilot collection intervals and total time, deciding on weekly collections over a month period.
There were some technical issues to decide also. From the beginning we wanted to extract DNA from the whole plastic, not only swabbing it, so we could sample any bacteria that could have burrowed its way into the plastic or attached so strongly that could not be removed easily. As for plastic types, we went for floating types: #2 can be found in many different forms, but we decided to go for the clear produce kind bags. For #4 we chose a white plastic bag, and #5 a yogurt container. Each came with its own pros and cons: #2 was the hardest to manipulate but the easiest to extract DNA from. For #5, we had to resort to cut the material in small pieces for the extraction.
First concern was the amount of DNA. It was clear that the longer the time, the more DNA we could extract from the plastic, as expected. That said, even samples with very low DNA content provided enough reads to be useful. We were happy to see that the sterilization method work, as non-deployed sterilized plastic resulted in very low DNA amounts, and when sequenced, in completely different microbial populations compared to the samples. To rule out contamination during extraction, we also ran “kit only” controls, which came out similar to the negative controls. Finally, we ran 2 samples of each, resulting in extremely similar results except one of the #5 plastic, which presented a much higher proportion of photosynthetic bacteria. We ascribe that to the fact that in the pilot run, the cages were too close to each other and one sample may have had more exposure to sunlight.
