Dneasy power water extraction kit

Snow was molten at 4°C, filtered through a 0.2 μm polycarbonate filter (47 mm, Isopore) and then stored frozen at −20°C until DNA extraction, performed with the DNeasy Power Water extraction kit (QIAGEN) following the protocol provided with the kit.
From the filters DNA was also extracted using a DNeasy Power Water extraction kit (QIAGEN) using an adapted protocol described in Dommergue et al. (2019) (link) to remove DNA from quartz. Amplification, library prep (MiSeq Illumina sequencing, 2 × 250 bp, Nextera XT Library Preparation Kit) and sequencing was carried out at the Environmental Microbial Genomics group at the Laboratoire Ampère (ECL Lyon, University of Lyon, France). Community diversity was targeted: the V3-V4 region of the bacterial 16S rRNA SSU gene was amplified using 341F/785R primers (S-D-Bact-0341-b-S-17/S-D-Bact-0785-a-A-21, Klindworth et al., 2013 (link)) and the fungal internal transcribed spacer (ITS) regions were amplified with primer pair 5.8S_Fung/ITS4 targeting the ITS2 region (Taylor et al., 2016 (link)).

The molten snow and water samples (1 L each) were filtered through a 0.2 μm polycarbonate filter (47 mm, Isopore). DNA was extracted from the filters using DNeasy Power Water extraction kit (Qiagen) following the provided protocol. Amplification, library preparation and sequencing was done at the Environmental Microbial Genomics group at the Laboratoire Ampère (ECL Lyon, University of Lyon, France). Community diversity was targeted: the V3–V4 region of the bacterial 16S rRNA SSU gene was amplified using 338F/518R primers and the fungal internal transcribed spacer (ITS) regions were amplified with primer pair ITS2-ITS4. Raw sequences were stored at NCBI BioProject database under Project ID PRJNA534428.

For enumeration of V. parahaemolyticus and V. vulnificus, a three-tube MPN was conducted, where 10 mL of seawater was first inoculated into 100 mL of APW. Then, 1 mL of seawater was added to three tubes of 9 mL of APW and one 9 mL tube of PBS to obtain a 10⁻¹ dilution. Serial dilutions and MPN methods were conducted using the procedure stated in the previous section. In addition, 500 mL of seawater was filtered using a 0.22 μm Sterivex filter (Manufacturer, City, State). The inoculated filters were then stored at −80°C until thawed for DNA extraction. DNA extractions were done using a DNeasy Power Water Extraction Kit (Qiagen, Germantown, MD, United States), following the manufacturer’s instructions.