Pre-isolation procedures matter–Comparison of different filtration methods prior to DNA isolation in river microbiome analysis

Abstract

A common method of preparing water samples for environmental DNA isolation is to vacuum filter the water sample through membrane filters. The aim of research was to test the performance of five methods of preparing river water samples for DNA isolation. DNA was isolated using a commercial kit and next-generation sequencing was performed on an Illumina Miseq platform. Pseudomonas spp. was the dominant bacterial genus in all samples. However, its relative abundance varied between samples. Depending on the volume of water filtered, the eluates yielded 62–63 % (V = 100 mL), 59–63.9 % (V = 50 mL), 17.8–19.4 % (V = 500 mL) of the relative abundance of Pseudomonas sp. In contrast, DNA isolation from the membrane filter (V = 100 mL) yielded 38 % and from the sediment after centrifugation 27 %. Differences were observed for all taxa obtained. The results indicate that even the sample volume used for filtration influences the results obtained from next-generation sequencing.