Optimizacija izolacije bakterijske genomske DNA za sekvenciranje s tehnologijo nanopore in analiza zaporedij izbranih sevov

Lovše, Urša (author), Lapanje, Aleš (mentor), Dolinar, Marko (comentor)

Hitro razvijajoče se tehnologije sekvenciranja, kamor uvrščamo tudi sekvenciranje s tehnologijo nanopore, povečujejo potrebo po razvoju novih učinkovitih metod izolacije bakterijske genomske DNA. Prednosti omenjene tehnologije so poleg cenovne dostopnosti, relativno hitrega in natančnega določanja zaporedij tudi sposobnost sekvenciranja posameznih molekul brez predhodnega pomnoževanja DNA. Da pa je sekvenciranje z omenjeno tehnologijo možno, je potrebno za pripravo genomskih knjižnic in nadaljnje sekvenciranje zagotoviti zadostno začetno koncentracijo molekul DNA s čim bolj enakomerno dolžino izoliranih fragmentov DNA. S tem si namreč v nadaljevanju dela, ki zajema bioinformacijsko analizo, med drugim olajšamo sestavljanje zaporedij genomov de novo. S tem dobimo vpogled v informacije o bakterijah, ki se odražajo v njihovih fenotipskih lastnostih, kot sta to na primer protimikrobna aktivnost in razgradnja lignina, ki sta bila tudi objekta naših raziskav. Tekom magistrskega dela smo sprva razvili metodo izolacije bakterijske genomske DNA za namen sekvenciranja z napravo MinION, na novo razvito metodo pa smo nato uporabili za izolacijo genomske DNA iz različnih sevov bakterij. Ustreznost omenjene metode za namen karakterizacije mikrobnih genomov smo preverili z bioinformacijsko analizo desetih sevov, izoliranih iz dveh zelo različnih okolij, pri čemer je prvo okolje predstavljala ustna votlina zdravih posameznikov (i) in drugo okolje razpadajoči ligninski material invazivnih rastlin (ii). Pri tem smo preverjali, ali so podatki, pridobljeni s to metodo, dovolj kvalitetni, da bomo z njimi lahko analizirali genome na treh ravneh kompleksnosti. Tekom magistrskega dela smo iz nukleotidnih zaporedij sevov, izoliranih iz okolja (i), identificirali več zapisov za sintezo sekundarnih metabolitov s potencialnim protimikrobnim učinkom proti bakteriji Aggregatibacter actinomycetemcomitans, pri sevih, izoliranih iz okolja (ii), pa smo identificirali zapise za encime s potencialno ligninolitično aktivnostjo. Poleg tega smo uspeli iz zaporedij, pridobljenih z na novo razvito metodo, pridobiti vpogled tudi v metabolne poti sevov, izoliranih iz omenjenih okolij.

• Language: Slovenian
• Keywords: sekvenciranje tretje generacije, tehnologija nanopore, bakterijska genomika, izolacija DNA, bioinformatika
• Work type: Master's thesis/paper
• Typology: 2.09 - Master's Thesis
• Organization: FKKT - Faculty of Chemistry and Chemical Technology
• Year: 2023
• PID: 20.500.12556/RUL-144389
• COBISS.SI-ID: 142822659
• Publication date in RUL: 17.02.2023

Secondary language

• Language: English
• Title: Optimisation of bacterial genomic DNA isolation for nanopore sequencing and sequence analysis of selected strains

Abstract

Rapidly developing sequencing technologies, including nanopore sequencing, increase the need to develop new efficient methods for isolating bacterial genomic DNA. The advantages of nanopore sequencing are affordability, relatively fast and accurate sequence acquisition and the ability to sequence individual molecules without prior DNA amplification. However, for sequencing with the aforementioned technology to be possible, it is necessary to ensure a sufficient initial concentration of DNA molecules whose length of isolated DNA fragments is as uniform as possible for the preparation of genomic libraries and further sequencing. In this way we facilitate the assembly of genomes de novo, which includes bioinformatic analysis. With the assembled genomes we gain insight into bacterial information, that is reflected in their phenotypic properties, such as antimicrobial activity and lignin degradation, which were the objects of our research. During the master thesis we initially developed a new method for bacterial genomic DNA isolation for sequencing with the MinION device and applied the newly developed method for genomic DNA isolation from different bacterial strains. For the purpose of verifying characterization of microbial genomes, we verified the adequacy with bioinformatic analysis of ten strains from two very different environments, the first being the oral cavity of healty individuals (i) and the second one the decomposing lignin material of invasive plants (ii). In this process we checked whether the data obtained with this method is of sufficient quality to be able to analyze genomes at three levels of complexity. In the course of master thesis we identified several records which could be linked to the synthesis of secondary metabolites with a potential antimicrobial effect against the bacterium Aggregatibacter actinomycetemcomitans from the sequences of strains isolated from the environment (i), and in the case the strains isolated from the environment (ii) we identified genes, coding for enzymes with potential ligninolytic activity. In addition from the obtained sequences we also managed to gain insight into the metabolic pathways of strains isolated from the aforementioned environments.

• Keywords: third generation sequencing, nanopore sequencing, bacterial genomics, DNA isolation, bioinformatics