Nanopore DNA sequencing with a sub-nm stop-and-go mechanical positioning system and current blockades or fluorescent tags

DNA sequencing using nanopores and a mechanical positioning system with 0.1-0.15 nm movement precision and ~1 μs movement times is proposed. A single strand of DNA (ssDNA) is attached to a homonucleotide header immobilized on a glass slide mounted on a platform in the cis chamber of an electrolytic cell (e-cell) with a nanopore. The header-strand is moved into the trans chamber and retracted mechanically one nucleotide at a time. Two approaches to sequencing are described. In the first the blockade current due to the segment of the header-strand in the pore is measured with a low bandwidth (< 1 KHZ); a Markov-like method identifies the next base from earlier blockade current levels. In principle, with a bandwidth of 1 KHz and an array of ~600 pores with a CMOS detector array a human genome (3 × 109 bases) can be sequenced in < 1.5 hours, or ~5 hours start-to-finish (sample preparation, ligation with header, threading, data acquisition, and post-processing, plus machine-learning-based methods for base calling). This theoretical sequencing speed is comparable to the practical speed of the recently reported Sequencing By Expansion (SBX) method; it remains to be seen if this level of performance can be delivered in practice. The second approach is based on replacing the enzyme motor in the Sequencing-by-Synthesis (SBS) method with a mechanical positioning system and using a header-strand with a hybridized primer. Synthesis consists of elongating the latter with a protected dNTP carrying a fluorescently tagged base that complements the next open base in the strand followed by deprotection of the dNTP and cleaving of the tag, the latter is detected optically with a Total Internal Reflection Fluorescence (TIRF) camera in trans. This electromechanical-optical alternative to SBS provides better control over sequencing by shielding earlier added nucleotides and reducing cross-talk in the neighborhood of the terminal nucleotide being extended. In theory, with a 1000 × 1000 array of DNA header-strands each ~200 bases long and a cycle time of ~90 seconds, a human chromosome can be sequenced in ~5 hours. The possibility of using the method for data retrieval from DNA-based storage is discussed.