Genotipizacija lokusa glavnog sustava tkivne podudarnosti srne, Capreolus capreolus (Linnaeus, 1758), sekvenciranjem nove generacije

master's thesis

• Title (english): Genotyping of the roe deer, Capreolus capreolus (Linnaeus, 1758), major histocompatibility complex by next generation sequencing
• Author: Mihaela Šurina
• Mentor: Ana Galov (mentor)
• Mentor: Elena Bužan (mentor)
• Committee member: Ana Galov (predsjednik povjerenstva)
• Committee member: Maja Matulić (član povjerenstva)
• Committee member: Duje Lisičić (član povjerenstva)
• Committee member: Sandra Hudina (član povjerenstva)
• Granter: University of Zagreb; Faculty of Science; (Department of Biology); Zagreb
• Defense date and country: 2022-01-26, Croatia
• Scientific / art field, discipline and subdiscipline: NATURAL SCIENCES; Biology

Abstract

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.

Abstract (english)

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.

• Keywords: NGS; MHC; Ion Torrent; AmpliSAT; priprema knjižnice
• Keywords (english): NGS; MHC; Ion Torrent; AmpliSAT; library preparation
• Language: croatian
• URN:NBN: urn:nbn:hr:217:548775
• Study programme: Title: Molecular Biology; Study programme type: university; Study level: graduate; Academic / professional title: magistar/magistra molekularne biologije (magistar/magistra molekularne biologije)
• Type of resource: Text
• File origin: Born digital
• Access conditions: Open access; Embargo expiration date: 2024-01-26
• Created on: 2022-02-07 14:03:58