Genotyping of the roe deer, Capreolus capreolus (Linnaeus, 1758), major histocompatibility complex by next generation sequencing

Geni glavnog sustava tkivne podudarnosti (eng. major histocompatibility complex, MHC) jedni su od najvažnijih biomarkera za procjenu genetičke raznolikosti populacija jer su izrazito varijabilni. Zbog uloge receptora MHC u vezanju i prikazivanju antigena novonastale mutacije omogućuju otpornost jedinki na veći broj patogena i rezultiraju velikim brojem gena i alela. Probleme u genotipizaciji predstavljaju višestruka polimorfna mjesta u alelima te je uz tradicionalnu metodu sekvenciranja po Sangeru potrebno raditi predkorak molekularnog kloniranja kako bi se razdvojile alelne faze. Metode sekvenciranja nove generacije (eng. next generation sequencing, NGS) omogućuju zasebno, a istovremeno sekvenciranje velikog broja fragmenata DNA te uvelike pojednostavljuju i ubrzavaju sam proces. Primjenila sam jednu od metoda NGS, Ion Torrent S5, u genotipizaciji lokusa DRB sustava MHC srne (Capreolus capreolus) kao primjera nemodelnog kralježnjaka. Umnožila sam egzon 2 lokusa DRB 14 uzoraka i napravila knjižnicu fragmenata za sekvenciranje. Dobivena očitanja analizirala sam u programskom paketu AmpliSAT i identificirala sam osam alela od kojih je pet poznatih, a tri su novootkrivena. Budući da u srna postoji samo jedan lokus DRB te su očitanja svakog uzorka bila izrazito velike pokrivenosti rezultati su bili jasni i jednoznačni. Primjenjivost tehnologije Ion Torrent potrebno je dalje testirati na uzorcima s udvostručenim lokusima te usporediti s rezultatima drugih metoda sekvenciranja.Genes of the major histocompatibility complex (MHC) are one of the most important biomarkers for assessment of genetic diversity of populations owing to their remarkable polymorphism. Due to role of MHC receptors in antigen binding and presentation, new mutations allow for resistance to a larger number of pathogens and result in a vast amount of genes and alleles. Multiple polymorphic sites in alleles, on the other hand, pose problems for successful genotyping by traditional methods and require application of molecular cloning in addition to Sanger sequencing in order to resolve allele phases. Next generation sequencing methods (NGS) have made possible sequencing of many DNA fragments individually, but in massive parallel fashion, thereby simplifying and speeding up the whole procedure. In this study I applied one of the NGS technologies, Ion Torrent S5, in genotyping MHC DRB locus of roe deer (Capreolus capreolus) as one of the nonmodel vertebrates. I amplified second exon of DRB locus in 14 individuals and constructed a sequencing library. I used online program AmpliSAT for data analysis and I identified eight alleles, five of which are already known and three that are new. Since roe deer has single DRB locus and sequencing yielded reads with great coverage, analysis was simple and straightforward. Applicability of Ion Torrent in genotyping needs to be further evaluated in species with duplicated DRB loci and in comparison with results of different sequencing technologies