Primerjava tehnologije DNA-mikromrež in sekvenciranja cDNA z nanoporami pri analizi transkriptoma celične linije HepG2 divjega tipa in z izbitim genom SC5D

Vičič, Žiga (author), Rozman, Damjana (mentor)

Potreba po razumevanju vpliva transkriptoma na celični fenotip v kliničnem okolju spodbuja razvoj novih generacij tehnologij določevanja nukleotidnega zaporedja prepisov. Prva tehnologija, ki je omogočila sočasno detekcijo izražanja velike množice genov, je tehnologija DNA-mikromrež. Nasledila jo je metoda direktnega sekvenciranja RNA, imenovana RNA-Seq, ki so jo omogočile tehnologije sekvenciranja naslednje generacije. Tehnologija sekvenciranja z nanoporami omogoča sekvenciranje celotnih prepisov, s čimer olajša bioinformacijsko analizo transkriptoma. Zaradi tradicionalne uporabe v medicinski diagnostiki se je metoda obratne transkripcije in kvantitativne verižne reakcije s polimerazo (RT-qPCR) uveljavila kot »zlati standard« za kvantifikacijo ravni izražanja posameznih genov. V okviru magistrske naloge smo primerjali rezultate analize diferenčno izraženih genov med celično linijo HepG2 divjega tipa in z izbitim genom SC5D, ki so bili zajeti z DNA-mikromrežami in s sekvenciranjem z nanoporami. Ravni izražanja izbrane skupine genov smo določili tudi z RT-qPCR in jih primerjali z rezultati ostalih dveh metod. Pri tem smo s sekvenciranjem z nanoporami v celični liniji z izbitim genom SC5D določili statistično pomembno spremembo v izražanju nekaterih genov, ki je pred tem nismo zaznali z analizo z mikromrežami. Pri izračunu korelacije med tehnologijama in rezultati meritev spremembe izražanja, določene z RT-qPCR, smo potrdili, da sta metodi primerljivi. Ob tem je bila korelacija z rezultati analize z mikromrežami presenetljivo višja. Prvič smo za statistično pomembno izražene gene, zaznane tako z analizo z mikromrežami kot s sekvenciranjem z nanoporami, izračunali korelacijo vrednosti dvojiškega logaritma faktorja spremembe. Ta je višja od v literaturi opisanih primerjav med tehnologijami sekvenciranja s sintezo in analizo z mikromrežami. Tekom magistrske naloge smo razvili cevovod za bioinformacijsko analizo, primerjali orodja za določitev diferenčnega izražanja genov in uspešno izvedli obogatitveno analizo skupin genov. Rezultati slednje bodo v prihodnje služili za razvoj nadaljnjih hipotez o vlogi izbitega gena SC5D v fenotipu celične linije.

• Language: Slovenian
• Keywords: analiza z mikromrežami, sekvenciranje z nanoporami, bioinformatika
• Work type: Master's thesis/paper
• Typology: 2.09 - Master's Thesis
• Organization: FKKT - Faculty of Chemistry and Chemical Technology
• Year: 2022
• PID: 20.500.12556/RUL-139147
• COBISS.SI-ID: 123539203
• Publication date in RUL: 31.08.2022

Secondary language

• Language: English
• Title: Comparison of DNA-microarray technology and nanopore cDNA sequencing in transcriptome analysis of HepG2 wild-type and SC5D gene knockout cell line

Abstract

The need for understanding the influence of the transcriptome on the phenotype of the cell in a clinical setting has governed the development of new generations of sequencing technologies. The first technology to enable a simultaneous detection of expression of a set of genes were DNA-microarrays. It was followed by the development of direct sequencing of RNA, called RNA-Seq, which was made possible by the next-generation sequencing technologies. Nanopore sequencing technology allows for the sequencing of whole transcripts, thus facilitating the bioinformatic analysis of the transcriptome. Due to its established role in the medical diagnostics, the method of reverse transcription and quantitative polymerase chain reaction has established itself as the “golden standard” of quantifying the expression level of individual genes. As part of the master’s thesis, we compared the results of the analysis of differentially expressed genes between the HepG2 wild-type and the SC5D knockout cell line, which were detected by DNA-microarrays and nanopore sequencing. The expression was also measured by RT-qPCR and compared with the results of the other two methods. By nanopore sequencing, we determined a statistically significant change in expression of several genes, which was not previously detected by microarray analysis. In calculating the correlation between the technologies and the results of the change in gene expression determined by RT-qPCR, we confirmed that the two methods are comparable. At the same time, the correlation with the results of the microarray analysis was surprisingly higher. We also report that we have for the first time determined the correlation between the values of the binary logarithm of the fold change in expression for statistically significantly expressed genes determined by both the microarrays and nanopore sequencing. The correlation factor is higher than the comparisons between the sequencing technologies using the sequencing by synthesis approach and microarrays that are described in the literature. During the master’s thesis, we developed a pipeline for bioinformatics analysis, compared tools for determining the differential expression of genes and successfully performed the gene set enrichment analysis. These will help develop further hypotheses on the role of the knocked-out gene SC5D in the cell phenotype.

• Keywords: microarray analysis, nanopore sequencing, bioinformatics