Sekvenciranje mitohondrijskega genoma uporabo CRISPR/Cas9 in tehnologije sekvenciranje DNA z nanoporo

magistrsko delo

Barbara Slapnik (Author), Maruša Debeljak (Mentor), Marko Dolinar (Thesis defence commission member), Aljaž Gaber (Thesis defence commission member), Vera Župunski (Co-mentor)

Abstract

Genetske spremembe v mitohondrijskem genomu so pomemben dejavnik pri razvoju mitohondrijskih bolezni. Te uvrščamo v skupino heterogenih bolezni, za katere je značilno pomanjkanje celične energije. Priprava knjižnice za sekvenciranje z uporabo CRISPR/Cas9 in tehnologija sekvenciranja DNA z nanoporo nam omogočata obogatitev mitohondrijskega genoma brez tarčnega pomnoževanja s PCR. Produkt takšnega sekvenciranja so dolgi odčitki (>10 kb), ki omogočajo sestavljanje mitohondrijskega genoma de novo in detekcijo večjih delecij ter ponovitvenih regij. Pri magistrskem delu smo dokazali, da lahko s tehnologijo CRISPR/Cas9 in sekvenciranjem z nanoporo obogatimo mitohondrijski genom in generiramo odčitke, ki pokrijejo celotno dolžino mitohondrijske DNA (mtDNA). Preverili smo različne bioinformacijske pristope, ki jih lahko uporabljamo pri analizi podatkov in rezultate med sabo primerjali za iskanje genetskih sprememb. Nato smo preverili zmožnosti tehnologije sekvenciranja z nanoporo za detekcijo nizkostopenjskih heteroplazmij, pri katerih celica vsebuje nizek delež mutirane mtDNA v primerjavi z mtDNA divjega tipa. Ugotovili smo, da lahko s to tehnologijo detektiramo do 10 % heteroplazmije. Detekcija heteroplazmije je pomembna pri diagnozi mitohondrijskih bolezni, saj lahko učinek ozkega grla med oogenezo in embriogenezo vodi do razvoja višje stopnje heteroplazmije, ki povzroči fenotipsko izražanje bolezni. Tehnologija sekvenciranja z nanoporo nam omogoča tudi identifikacijo modificiranih baz, ki so pomembne pri regulaciji izražanja genov. Pri analizi smo identificirali prisotnost 5-metilcitozinov na mtDNA in ugotovili, da je njihova frekvenca nizka. Uporaba zgoraj omenjenega pristopa bi omogočila globlje razumevanje mitohondrijske genetike in epigenetike ter posledično delovanja mitohondrija.

Keywords

mitohondrijski genom;mitohondrijske bolezni;heteroplazmija;epigenetika;sekvenciranje DNA z nanoporo;magistrska dela;

Data

Language:	Slovenian
Year of publishing:	2022
Typology:	2.09 - Master's Thesis
Organization:	UL FKKT - Faculty of Chemistry and Chemical Technology
Publisher:	[B. Slapnik]
UDC:	577.21(043.2)
COBISS:	120528899
Views:	60
Downloads:	20
Average score:	0 (0 votes)
Metadata:

Other data

Secondary language:	English
Secondary title:	Sequencing of mitochondrial genome with CRISPR/Cas9 technology and nanopore sequencing
Secondary abstract:	Genetic variants of mitochondrial genome are an important driver of mitochondrial disorder pathology. Mitochondrial disorders are heterogeneous diseases characterized by a lack of cellular energy. Using targeted nanopore sequencing in combination with CRISPR/Cas9 technology enables amplification-free sequence enrichment of mitochondrial DNA (mtDNA). Nanopore sequencing produces long reads (> 10 kb), which improves de novo assembly and detection of large sporadic deletions and tandem repeats. In this study, we demonstrated that this technology enriches the mitochondrial genome and generates full-length nanopore reads of mtDNA. We tested different bioinformatics approaches for analyzing the data and compared the results of these approaches for variant calling. We verified the ability of nanopore sequencing for the detection of low-level DNA heteroplasmy, where the cell contains a low proportion of mutated mtDNA molecules compared to wild-type mtDNA. We determined that nanopore sequencing is able to detect down to 10 % heteroplasmy. Detecting a low-heteroplasmic variance is crucial for the diagnosis of mitochondrial diseases, as the bottleneck effect during oogenesis and embryogenesis can lead to the sampling and subsequent expansion of rare mutant mtDNA. Nanopore sequencing technology is also able to detect modified bases that are important for epigenetic control of gene expression. We identified the presence of 5-methylcytosines on mtDNA and observed that their frequency is low. The application of the presented approach would provide deeper understanding of mitochondrial genetics, epigenetics and consequently, of mitochondrial biology.
Secondary keywords:	mitochondrial genome;mitochondrial diseases;heteroplasmy;epigenetics;nanopore sequencing;Mitohondriji;DNA;Univerzitetna in visokošolska dela;
Type (COBISS):	Master's thesis/paper
Study programme:	1000377
Embargo end date (OpenAIRE):	1970-01-01
Thesis comment:	Univ. v Ljubljani, Fak. za kemijo in kemijsko tehnologijo, smer Biokemija
Pages:	69 str.
ID:	16207868