Targeted Enrichment and Nanopore Sequencing of RNA Transcripts from Breast Cancer Risk Genes
Chang, Jessie Jie-Youen
Cite this item:
Chang, J. J.-Y. (2018). Targeted Enrichment and Nanopore Sequencing of RNA Transcripts from Breast Cancer Risk Genes (Thesis, Bachelor of Biomedical Sciences with Honours). University of Otago. Retrieved from http://hdl.handle.net/10523/8601
Permanent link to OUR Archive version:
http://hdl.handle.net/10523/8601
Abstract:
Aberrant alternative splicing of hereditary breast and ovarian cancer genes has been associated with an increased susceptibility for breast and/or ovarian cancer. However, studying the effects of gene variants on splicing patterns proves to be difficult for classifying Variants of Uncertain Significance (VUS) – variants which lack the evidence to be categorised as benign or pathogenic. This is particularly the case for the BRCA1 gene, which is poorly expressed, with a long transcript length.
In this study, a novel RNA enrichment technique using the Oxford Nanopore MinION long-read sequencer was developed and tested to alleviate these limitations. The main hypotheses were that direct RNA sequencing can identify full-length splice isoforms of genes of interest, and that these results can be enhanced using a novel gene enrichment technique.
Total RNA was extracted from a control lymphoblastoid cell line and reverse-transcribed into cDNA. Second-strand cDNA synthesis was carried out by incorporating a T7-polymerase binding site attached to a gene-specific primer for the genes of interest. In vitro transcription was carried out with T7-polymerase to enrich the RNA isoforms, which were then sequenced with the Direct RNA Sequencing Kit (Oxford Nanopore Technologies, UK). Several bioinformatic pipelines were evaluated, as bespoke pipelines for analysing this novel dataset were unavailable. The final pipeline consisted of alignment to the Human Genome Reference Build 38 with Minimap2, conversion to BAM files, sorting and indexing with Samtools. The output file was visualised on the Integrative Genomics Viewer, and the reads were manually counted and processed in Excel.
The results exhibited evidence of transcript enrichment for three genes of interest (BRCA1, RAD51C, CHEK2) and a control gene (GAPDH). Of note, GAPDH presented isoform abundance ratios which were similar in three separate experiments, indicating potentially quantitative and reproducible linear enrichment. Full-length isoforms of GAPDH, RAD51C and BRCA1 were able to be sequenced with the Direct RNA Sequencing Kit. Additionally, novel isoforms which have not been previously described for RAD51C were detected, which include Δ 3,5-6, Δ 3,7 and Δ 4,7.
Novel methodologies and proof-of-principle experiments were executed to enrich and analyse specific transcripts using direct RNA nanopore sequencing. The enrichment method should be further validated with other approaches such as RT-qPCR and optimised for use with relevant genes. Provided that a bespoke pipeline for long direct RNA sequencing becomes available, this novel method may provide a powerful new approach for interpreting the clinical significance of variants of uncertain significance which may impact on splicing patterns.
Date:
2018
Advisor:
Kennedy, Martin; Walker, Logan; Miller, Allison; Cree, Simone; Pearson, John; Lattimore, Vanessa
Degree Name:
Bachelor of Biomedical Sciences with Honours
Degree Discipline:
Department of Pathology and Biomedical Science, UOC
Publisher:
University of Otago
Keywords:
Nanopore; Direct RNA Sequencing; RNA Enrichment; Lymphoblastoid Cell Line
Research Type:
Thesis
Languages:
English
Collections
- Pathology - Christchurch [76]
- Thesis - Honours [340]