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dc.contributor.advisorKennedy, Martin A
dc.contributor.advisorWalker, Logan C
dc.contributor.authorde Jong, Lucy Clair
dc.date.available2017-05-30T22:04:18Z
dc.date.copyright2016
dc.identifier.citationde Jong, L. C. (2016). Nanopore Sequencing of RNA from Breast Cancer Genes (Thesis, Bachelor of Biomedical Sciences with Honours). University of Otago. Retrieved from http://hdl.handle.net/10523/6936en
dc.identifier.urihttp://hdl.handle.net/10523/6936
dc.description.abstractAbnormal mRNA splicing can disrupt gene function and influence the course of disease. Analysis of abnormal splicing is an important part of determining whether a particular genetic variant found in the population is pathogenic or not. However, to correctly identify abnormal splicing, we must first understand what is normal. This project assessed the isoforms of the genes BRCA1 and BARD1, which are particularly relevant to the onset of breast cancer. BRCA1 is a tumour suppressor gene implicated in breast cancer onset. BARD1 codes for a protein that interacts with BRCA1 and produces a smaller mRNA transcript. Normal exon skipping events have been identified for both BRCA1 and BARD1, however, current methods are unable to reliably identify full transcripts. This has resulted in knowledge of individual exon skipping events but often does not tell us whether multiple events occur in the same transcript. The MinION nanopore sequencer (Oxford Nanopore Technologies), uses a nanopore to produce long-read, single molecule sequences. This has great potential for identifying multiple long isoforms, which is not practical using current technologies. The aim of this project was to examine the ability of the MinION to identify mRNA splicing patterns of transcripts derived from BRCA1 and BARD1. All mRNA from a normal lymphoblastoid cell line was converted to cDNA and targeted genes of interest were amplified by polymerase chain reaction (PCR). All potential isoforms generated from BRCA1 and BARD1 were then pooled and analysed using the MinION sequencer. After trialling many different analysis methods, the read data was analysed using the BLAST-like Alignment Tool (BLAT) with two outputs, a tabular and a graphical format. The tabular format grouped reads into potential isoforms, while the graphical format allowed visualisation of these isoforms and identified the exon/intron boundaries. Using both these formats 34 BRCA1 isoforms and 39 BARD1 isoforms were identified, 24 and 17 of which were potential novel isoforms respectively. Two of these novel isoforms from the BRCA1 dataset (Δ10-17 and Δ11q21) were further verified using Sanger sequencing. This was a proof of principle research project that demonstrated the potential use of the MinION nanopore sequencer for successful characterisation of multiple mRNA isoforms. This research has successfully identified a number of novel isoforms from the BRCA1 and BARD1 genes using the MinION sequencing device.
dc.format.mimetypeapplication/pdf
dc.language.isoen
dc.publisherUniversity of Otago
dc.rightsAll items in OUR Archive are provided for private study and research purposes and are protected by copyright with all rights reserved unless otherwise indicated.
dc.subjectNanopore
dc.subjectlong-read
dc.subjectBRCA1
dc.subjectRNA
dc.subjectisoforms
dc.subjectBARD1
dc.titleNanopore Sequencing of RNA from Breast Cancer Genes
dc.typeThesis
dc.date.updated2016-11-15T22:46:13Z
dc.language.rfc3066en
thesis.degree.disciplinePathology (UOC)
thesis.degree.nameBachelor of Biomedical Sciences with Honours
thesis.degree.grantorUniversity of Otago
thesis.degree.levelHonours
otago.openaccessOpen
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