Current high throughput/short read sequencing technologies do not resolve all long repetitive sequences present in both the human genome and present in some human pathogens. These sequences are mechanistically important in disease and therapeutics (e.g. mitochondrial disease, autoimmune disease, neurological disease, cancer, and antibiotic resistance). I will build on the last 3 years of our improvements in MinION sequencer performance to significantly improve sequencing read length. In year one, I will develop a rapid, simple, sensitive, and accurate method for human mitochondria genotyping using full-length mitochondrial genome MinION reads (~16.6 Kb). In year two, I will extend these approaches to develop a method for sequencing of up to 1 mega base long reads of genomic DNA with the MinION. These advances change the current paradigm for sequencing difficult repetitive sequences, in which, 20 kb is viewed as a long read. Longer read lengths (to mega bases in length) are needed to resolve some of the genomic gaps in difficult to sequence regions (e.g. centromere, and satellite DNA). In our laboratory, I will use these advances to next investigate the role of adducted mitochondrial DNA bases in aging and disease. The mega-base long read technology of year two will be used in our lab for a planned collaboration to sequence the centromere region of pediatric leukaemias believed to result from Robertsonian translocation of chromosomes 13 and 14 (i.e. (rob13;14)c). These technology improvements will have widespread application in both mitochondrial research and genome sequencing for personalized medicine.
Current high throughput/short read DNA sequencing technologies do not resolve a number of long repetitive DNA sequences in human genomes and in human pathogens. These sequences are critical to understanding aging, disease (e.g. cancer, neurological disorders, autoimmune diseases), and antibiotic resistance. We will develop a low cost, simple, long read sequencing methodology to resolve many of these critical sequences using the MinION nanopore sequencer.
