Comparison of MHC genotyping methods in the genome with duplicated locus

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