RESUMO
Nanopore-based DNA sequencing is the most promising third-generation sequencing method. It has superior read length, speed, and sample requirements compared with state-of-the-art second-generation methods. However, base-calling still presents substantial difficulty because the resolution of the technique is limited compared with the measured signal/noise ratio. Here we demonstrate a method to decode 3-bp-resolution nanopore electrical measurements into a DNA sequence using a Hidden Markov model. This method shows tremendous potential for accuracy (~98%), even with a poor signal/noise ratio.
Assuntos
Algoritmos , DNA/genética , Nanoporos , Análise de Sequência de DNA/métodos , Sequência de Bases , Eletricidade , Genoma Humano/genética , Humanos , Cadeias de Markov , Razão Sinal-RuídoRESUMO
Nanoscale pores have proved useful as a means to assay DNA and are actively being developed as the basis of genome sequencing methods. Hairpin DNA (hpDNA), having both double-helical and overhanging coil portions, can be trapped in a nanopore, giving ample time to execute a sequence measurement. In this article, we provide a detailed account of hpDNA interaction with a synthetic nanopore obtained through extensive all-atom molecular dynamics simulations. For synthetic pores with minimum diameters from 1.3 to 2.2 nm, we find that hpDNA can translocate by three modes: unzipping of the double helix and--in two distinct orientations--stretching/distortion of the double helix. Furthermore, each of these modes can be selected by an appropriate choice of the pore size and voltage applied transverse to the membrane. We demonstrate that the presence of hpDNA can dramatically alter the distribution of ions within the pore, substantially affecting the ionic current through it. In experiments and simulations, the ionic current relative to that in the absence of DNA can drop below 10% and rise beyond 200%. Simulations associate the former with the double helix occupying the constriction and the latter with accumulation of DNA that has passed through the constriction.