Crayfish play an important role in freshwater ecosystems and are among the most successful invasive species, yet their pathogens, including viruses, remain poorly studied. In this master’s thesis, we investigated the virome of the invasive signal crayfish (Pacifastacus leniusculus) and the effect of different methods of hepatopancreas sample preparation on the detection of viral sequences using high-throughput sequencing. Samples were collected at four locations along the invasion gradient in the Korana River. We compared the effects of two RNA isolation methods, using the RNeasy Lipid Tissue Mini Kit (MiniKit) and TRIzol™ reagent, two different homogenization matrices, and library preparation with and without ribosomal RNA (rRNA) depletion. In addition, viral particles were concentrated by ultracentrifugation in a subset of samples. After sequencing on the Illumina platform, the data were analysed using bioinformatic approaches that included quality control, de novo assembly, taxonomic identification, and phylogenetic analyses. The results showed that sample preparation affected the detection of viral sequences, with rRNA depletion increasing their relative proportion in the sequencing data. In our case, ultracentrifugation did not improve the detection of viral material. We detected sequences of known signal crayfish viruses as well as novel picorna-like viral sequences. Signal crayfish-associated toti-like virus 1 (SCaTlV1) was confirmed at all locations, whereas signal crayfish-associated reo-like virus 1 (SCaRlV1) and signal crayfish-associated hepe-like virus 1 (SCaHlV1) were confirmed at three locations but not at the invasion front. The results indicate that the signal crayfish virome is diverse and that its composition differs depending on the sampling location, while the detection of viral sequences also depends on sample preparation.
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