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Geni glavnog sustava tkivne podudarnosti (eng. major histocompatibility complex, MHC) kodiraju MHC molekule sustava I i II čija je funkcija predočavanje antigena pri pokretanju adaptivnog imunosnog odgovora. Tijekom evolucije kod MHC gena događa se duplikacija lokusa koja povećava varijabilnost, a time i sposobnost obrane organizma. Duplikaciju lokusa karakterizira pojava više od dva alela po jedinci, koji se tijekom genotipizacije koamplificiraju s određenim parom početnica. Determinacija alela se, stoga, uz metodu Sangerovog sekvenciranja upotpunjuje metodom molekularnog kloniranja ili se genotipizacija temelji na metodama sekvenciranja nove generacije (eng. next generation sequencing, NGS). U ovom diplomskom radu usporedila sam tri metode genotipizacije MHC sustava na primjeru nemodelnog kralježnjaka obični jelen Cervus elaphus (Linnaeus, 1758). Genotipizirala sam egzon 2 DRB lokusa MHC sustava II u 10 uzoraka vrste obični jelen te dobivene razultate analizirala u računalnim programima Bioedit, Seqscape te računalnom paketu AmpliSAT. Kao najoptimalnija pokazala se metoda sekvenciranja zasnovana na sintezi (Illumina) kojom je identificirano najviše alela, njih 19. Sa sve tri metode identificiran je ukupno 21 alel, od čega 9 nije do sada bilo otkriveno. The genes of the major histocompatibility complex (MHC) encode MHC class I and II receptors whose function is to present the antigen when triggering an adaptive immune response. During the evolution of the MHC genes, duplication of loci takes place, which increases the variability and thus the ability of the organism to defend itself. Duplication of loci is characterized by the appearance of more than two alleles per individual, which during genotyping co-amplify with a particular pair of primers. Along with the method of Sanger sequencing supplemented by the molecular cloning method, genotyping is based on next generation sequencing (NGS) methods. In this thesis, I compared three methods of genotyping the MHC system on the example of the nonmodel vertebrate red deer, Cervus elaphus (Linnaeus, 1758). I genotyped exon 2 of the DRB locus of the MHC system II in 10 samples of the red deer species and analyzed the obtained results in the computer programs Bioedit, Seqscape and the computer package AmpliSAT. The sequencing by synthesis (Illumina) NGS method, which identified the most alleles (19) proved to be the most optimal. A total of 21 alleles were identified with all three methods, of which 9 have not been detected so far.

The presence of a novel adenovirus (AdV) was detected by PCR and sequencing, in the internal organs of a captive polar bear that had died in the Budapest zoo. The virus content of the samples proved to be high enough to allow for conventional Sanger sequencing on PCR-amplified genomic fragments. With this approach, the sequence of the entire genome of the putative polar bear adenovirus 1 (PBAdV-1) was obtained. Although the genome was found to be short, consisting of 27, 952 base pairs merely, with a relatively balanced G + C content of 46.3 %, its organisation corresponded largely to that of a typical mastadenovirus. Every genus-common gene could be identified except that of protein IX. The short E3 region of the PBAdV-1 consisted of two novel, supposedly type- specific ORFs only, whereas no homologue of any of the E3 genes, usually conserved in mastadenoviruses, such as for example that of the 12.5 K protein, were present. In the E4 region, only the highly conserved gene of the 34 K protein was found besides two novel ORFs showing no homology to any known E4 ORFs. In silico sequence analysis revealed putative splicing donor and acceptor sites in the genes of the E1A, IVa2, DNA-dependent DNA polymerase, pTP, 33 K proteins, and also of U exon protein, all being characteristic for mastadenoviruses. Phylogenetic calculations, based on various proteins, further supported that the newly- detected PBAdV is the representative of a new species within the genus Mastadenovirus, and may represent the evolutionary lineage of adenoviruses that coevolved with carnivorans.

Gen CYP2D6 spada u skupinu farmakogena koji sintetiziraju proteine uključene u apsorpciju, distribuciju, metabolizam i ekstrakciju (ADME) lijekova u organizmu. Ovaj gen smješten je na regiji 22q13.2, dugačak je 9,7 kb i kodira protein uključen u metabolizam antidepresiva, antipsihotika, neuroleptika, kao i lijekova koji sluţe za prevenciju kardiovaskularnih bolesti. U različitim se populacijama javljaju različite vrste genskih varijacija gena CYP2D6 s različitim učestalostima. Sekvenciranjem gena CYP2D6 na uzorku 323 hrvatskih Roma metodom sekvenciranja sljedeće generacije, kod 141 ispitanika otkrivene su nove mutacije. U ovom radu umnoţeni su dijelovi gena CYP2D6 prethodno okarakterizirani kao novootkrivene mutacije te je sekvenciranje ponovljeno Sangerovom metodom. PotvrĎena je mutacija na lokaciji chr22:42.129.127 (GRCh38) te je ustanovljena prevalencija od 0,159% u romskom uzorku. Preostale dvije mutacije nisu potvrĎene te je ustanovljena greška prilikom sekvenciranja metodom sljedeće generacije. Korištenjem softvera Protein Variant Effect Analyzer utvrĎeno je da se novootkrivena mutacija nalazi u protein-kodirajućoj regiji, no kako dovodi do sinonimne, odnosno tihe supstitucije, ovom promjenom nukleotida ne dolazi do zamjene aminokiseline odnosno do promjene strukture proteina. CYP2D6 gene belongs to a group of pharmacogens that synthesize proteins involved in the absorption, distribution, metabolism and extraction (ADME) of drugs. This 9.7 kb long gene is located in the 22q13.2 region and encodes the protein involved in the metabolism of antidepressants, antipsychotics, neuroleptics, and drugs used to prevent cardiovascular disease. Different populations show different genetic variants of the CYP2D6 gene with different frequencies. By sequencing the CYP2D6 gene on a sample of 323 Croatian Roma by next generation sequencing, new mutations were detected in 141 subjects. In this paper, the regions of the CYP2D6 gene in which new mutations were found, were amplified and sequencing was repeated by Sanger's method. Mutation was confirmed at location chr22:42.129.127 (GRCh38) with a prevalence of 0.159% in the Roma. Other two mutations were not confirmed, but an error that occurred during sequencing by the next generation method was established. Using the Protein Variant Effect Analyzer software, the newly discovered mutation is within the protein-coding region, but as a result of synonyms, ie silent substitution, this SNP does not result in the replacement of the amino acid or the change of the protein structure.