A Non-Traditional Approach to Whole Genome Ultra- Fast, Inexpensive Nanopore-Based Nucleic Acid Sequencing

DNA sequencing claims responsibility for breakthroughs in understanding the molecular basis of life, and improving quality of life through advances in prognosis, diagnosis, treatment, and cure of disease. The last 30 years have seen an exponential improvement onto the original Sanger sequencing by synthesis, as well as the emergence of new technologies. Still the mandate for cheaper, faster, longer, and more accurate reads hasn't been satisfied. We are proposing a single molecule approach by combining unassisted nanoporebased sequencing with labeled DNA, where the ion-channel readout of current vs. time (i-t) may represent base sequence. Pyrimidines on DNA are labeled selectively with Osmium tetroxide 2,2'-bipyridine (OsBp) ahead of sequencing. The OsBp label slows down the translocation to detectable levels, and provides base discrimination between labeled deoxythymidine, labeled deoxycytidine, and an intact base. This technology promises to sequence DNA with no limit in strand length, without amplification, and without the use of a processing enzyme; it requires consensus building, but no assembly and no scaffolding. To facilitate consensus building for a human chromosome long DNA, highly repetitive DNA sequences, such as the Alu repeats, may serve as markers. Observed translocation times of a series of osmylated oligos via the wt a-Hemolysin nanopore are exploited to estimate the time it takes (1 hour) to sequence a 100,000,000 bp genome at 128x coverage using one MinION™ device from Oxford Nanopore Technologies. This technology has the potential for mapping protein bound regions in dsDNA, sequencing RNA, as well as identifying methylated and other rare bases.