Metagenomic Analysis of Brine Shrimp in Saline Lakes in the Pavlodar Region
For viral purification and nucleic acid isolation, approximately 10 mg of Artemia cysts were collected from each of the 12 salty lakes and combined by regions, resulting in four composite pools representing the respective sampling regions. The pooled samples were homogenized using a TissueLyser (Qiagen) along with lysis buffer (Buffer RLT Plus, Qiagen) and 5 mm stainless-steel beads, ensuring thorough mixing through vigorous vortexing. Subsequently, the suspension underwent centrifugation at 10,000g using a benchtop microcentrifuge for 5 minutes at 4C to eliminate large cyst residues. This centrifugation step was repeated twice to ensure optimal separation. Additionally, a 0.45 m filter (Merck Millipore, MA, USA) was used to remove eukaryotic and some bacterial cell-sized particles from each sample pool. The DNase kits (Turbo DNase from Thermo Fisher, Baseline-ZERO from Epicentre, and Benzonase from Novagen) and RNase kits (Promega, Madison, WI, USA) were utilized to enzymatically degrade unprotected nucleic acid present in the filtrates that were enriched with viral particles. The residual viral RNA and DNA were subsequently isolated using the QIAamp viral RNA Minikit (Qiagen) in accordance with the protocol provided by the manufacturer. Four pooled samples were produced, each containing virus sequences of both DNA and RNA, subjected to reverse transcription (RT) and double-stranded DNA construction utilizing QIASeq Stranded RNA Library Kit (Qiagen, Germany) along with a random hexamer primer at a concentration of 100 pmol. Subsequently, four libraries were generated via the NEBNext Ultra DNA Library Prep Kit for Illumina (New England Biolabs, USA) and sequenced on the Miseq Illumina platform.