| Popis |
Ectoparasitic flatworms from the class Monogenea represent serious fish pathogens and their presence in stocks can lead to significant losses in fish host populations. Despite this fact, the running research has been focused mainly on morphological and phylogenetical characteristics of these worms and the information related to the biochemical and molecular nature of the physiological processes is rather sporadic, as well as the knowledge of the molecules produced by monogeneans and their role in host-parasite interaction. From a large group of Monogenea only a few monogenean protein molecules have been deeply molecularly/biochemically characterized, e.g. peptidases (cathepsins B, D and L) and their inhibitors (cystatins and serpins), thioredoxin and annexin. Up to this date only two monogenean species were deeply sequenced on whole-genomic level. Genomes of Gyrodactylus salaris (Monopisthocotylea: Gyrodactylidae) and Protopolystoma xenopodis (Polyopisthocotylea: Polystomatidae) are currently available in public sequence databases. We would like to change this situation and therefore we adopted Eudiplozoon nipponicum from second large group of Monogenea Polyopisthocotylea (Diplozoidae) as an experimental organism for purpose recognize the important physiological pathways and crucial protein molecules involved in interactions with the fish host. E. nipponicum is a blood-feeding parasite inhabiting gills predominantly of common carp Cyprinus carpio, which is economically very important fish species in the Czech Republic. We started with analyses leaded to generation of E. nipponicum genome, transcriptome and secretome database. For the first E. nipponicum genome draft we used three sequencing techniques. 454/Roche sequencing (Junior platform), Illumina sequencing (MiSeq and HiSeq platforms) and Oxford Nanopore sequencing (MinION platform). 165,871,596 genomic raw reads were generated in total and after quality processing (trimming, removing contaminants, errors correction, etc.), 130,741,241 reads were used for the draft of genome assembly. Currently, the mitochondrial genome was already reconstructed and further bioinformatic evaluation of obtained assembled data is in progress. Generating of transcriptomic/secretomic dataset was based on usage of two sequencing methods (454/Roche and Illumina MiSeq platform) and mass spectrometry analysis (HPLC MS/MS, Orbitrap Elite Hybrid spectrometer). Total RNA, in the form of cDNA, was sequenced and raw reads were processed and assembled using specific bioinformatic tools. Based on the statistical evaluation and post-filtering steps, final transcriptome database contains 37,062 contaminant-free transcripts with corresponding translated NUMBER proteins. During annotation step, 19,539 (52.7 %) transcripts were homologous to some record in selected public sequence databases and 18,556 (50.1 %) of them were linked with sequences related to organisms only in phylum Platyhelminthes deposited in UniProtKB/TrEMBL database. Excretory-secretory products (ESP), protein compounds present in parasite secretome, were obtained from ~100 living adults and analysis of secretome peptide mixture revealed 1,033 proteins.
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