Browsing by Author "Olander, Viktor Otto Julius"

Preserving viral nucleic acids is of outmost importance to capture the viral diversity in metagenomic studies. In my master’s thesis, I compare viromes of genus Culex mosquitos stored in two different virus storage media and empty tubes. The mosquitos were collected from Kalajärvi in Espoo, Finland in the summer of 2020 as larvae and were grown to adults in laboratory conditions. Eight pools of five female mosquitos each were stored in each of the two media as well as empty tubes and the samples were homogenized The homogenates were filtered, and the RNA was extracted from them with TRIzol reagent. RNA was then reverse-transcribed to cDNA and amplified with a whole transcriptome amplification kit. The PCR product was prepared with a library preparation kit for sequencing with Illumina Next Generation Sequencer. The resulting reads were processed with a bioinformatic pipeline for identifying viruses from metagenomic sequence data. The results show a clear difference in virus species distribution by storage media. We identified 34 virus species from at least 13 families. Samples stored in ∑-Virocult had the highest yield of viral reads (70.40% of all reads from the pools) as well as the widest variety of mosquito species (n=26). Samples stored in empty tubes had the second most mosquito species (n=10) but the lowest viral read yield (1.25%). RNAlater stored samples had the least virus species (n=7) but a higher viral read percentage than those stored in empty tubes (3.26%). The results indicate the importance of choice of storage media. Since ∑-Virocult had the highest amount of reads and the widest variety, it might be the most useful storage media for our purposes. However, some viruses were found in other samples but not in ∑-Virocult stored samples, indicating a need of different storages conditions for different viruses. It is also important to be consistent in the use of media as it may affect virome results. More work needs to be done to assess if these results are true for other mosquito species as well.