Dnbseq 400 platform

To obtain the virome, the samples were sequentially filtered through 0.4 and 0.2 µm polycarbonate filters (Sartorius, Göttingen, Germany; Reatrack-Filter, Obninsk, Russia) to remove detritus and zoo-, phyto-, and bacterio-plankton. The filtrates from each sample were concentrated with a tangential flow filtration VivaFlow 200 (Sartorius, Göttingen, Germany) to a volume of 100 mL, and then centrifuge concentrators (50 kDa) were used to further concentrate them to 1 mL, at 4 °C and 3000 rpm using VivaSpin Turbo 15 (Sartorius, Göttingen, Germany). The concentrate was frozen in liquid nitrogen and stored at −70 °C until further analysis.
Total RNA was isolated using ExtractRNA (Evrogen, Moscow, Russia) according to the manufacturer’s protocol. To prepare the RNA-seq library according to the MGIEasy RNAseq Library Prep Set protocol (MGI Tech, Shenzhen, China), 100–200 ng of isolated RNA was used. The following steps were performed: RNA fragmentation, reverse transcription, second chain synthesis, the polishing of dsDNA fragment ends, and adapter ligation (containing 10 nucleotide single-end indexes). Sequencing was run on the DNBSEQ-400 platform (MGI Tech, Shenzhen, China) with paired-end reads (2 × 150 bp).

Sequencing was performed by MGITECH DNBSEQ 400 platform (MGITECH, People’s Republic of China), which showed excellent results in benchmark metagenomic studies (27 (link)). For the sequencing DNBSEQ-G400RS High-throughput Sequencing Set (PE150) reagent kit was used, since it was the maximum readlength available at the time of experiments. Demultiplexing of indexed samples was performed on the instrument. Quality control was performed by the analysis of raw read quality data with seqkit (28 (link)) and FastQC tools (29 ).