ena-STUDY-ECOLE POLYTECHNIQUE FEDERALE DE LAUSANNE, SWITZERL-17-07-2019-09:18:52:640-695
Benchmarking protocols for the generation of viromes from stream biofilms
Here, we evaluate a range of methods to separate viral particles from stream biofilms, and to concentrate and purify them prior to DNA extraction and metagenome sequencing. Based on epifluorescence microscopy counts of viral-like particles (VLP) and DNA yields, we optimize a protocol including treatment with tetrasodium pyrophosphate and ultra-sonication to disintegrate biofilms, tangential-flow filtration to extract and concentrate VLP, followed by ultracentrifugation in a sucrose density gradient to isolate VLP from the biofilm slurry. Our protocol optimization provides an important step towards a better understanding of the ecological role of viruses in stream biofilms.
Here, we evaluate a range of methods to separate viral particles from stream biofilms, and to concentrate and purify them prior to DNA extraction and metagenome sequencing. Based on epifluorescence microscopy counts of viral-like particles (VLP) and DNA yields, we optimize a protocol including treatment with tetrasodium pyrophosphate and ultra-sonication to disintegrate biofilms, tangential-flow filtration to extract and concentrate VLP, followed by ultracentrifugation in a sucrose density gradient to isolate VLP from the biofilm slurry. Our protocol optimization provides an important step towards a better understanding of the ecological role of viruses in stream biofilms.