Increasing the accuracy of nanopore DNA sequencing using a time-varying cross membrane voltage

Abstract

Nanopore DNA sequencing is limited by low base calling accuracy. Improved base-calling accuracy has so far relied on specialized base-calling algorithms, different nanopores and motor enzymes, or biochemical methods to re-read DNA molecules. Two primary error modes hamper sequencing accuracy: enzyme mis-steps and sequences with indistinguishable signals. We vary the driving voltage across an MspA nanopore between 100 to 200 mV with a frequency of 200 Hz, changing how the DNA strand moves through the nanopore. As a DNA helicase moves the DNA through the nanopore in discrete steps, the variable voltage positions the DNA continuously between these steps. The resulting electronic signal can be analysed to overcome the primary error modes in base-calling. Single-passage de novo base-calling accuracy in our device increases from 62.7 ± 0.5% with a constant driving voltage to 79.3 ± 0.3% with a variable driving voltage. Our variable-voltage sequencing mode is complementary to other advances in nanopore sequencing and is amenable to incorporation into other enzyme-actuated nanopore sequencing devices.

Opportunities and challenges in long-read sequencing data analysis

“…Many modifications, including 5mC, do not influence the SMRT polymerase' dynamics sufficiently to be detected at a useful sensitivity (5mC requires 250× coverage). In this case, software improvements are unlikely to yield significant gains, and improvements in sequencing chemistries are probably required [168]. Nanopore sequencing appears more amenable to the detection of a wide array of base modifications (to date: 5mCG, BrdU, 6mA), but the lack of ground truth data to train models and the combinatorial complexity of introducing multiple alternative bases are hindering progress towards a goal of seamless basecalling from an extended alphabet of canonical and non-canonical bases.…”

A new method for long-read sequencing of animal mitochondrial genomes: application to the identification of equine mitochondrial DNA variants

“…The possibility to do so rapidly and at a relatively modest cost is of added interest, and may be of interest for field studies since the MinION sequencer is portable and able to perform real-time sequencing and base-calling (46). At present, the technology is still hindered by the limited level of sequence accuracy but this is rapidly changing as sequencing chemistry and base-calling algorithms are evolving (47,48). The variety of flow cells and equipment available enables to better tailor the sequencing tools according to the number of samples.…”

Actinoporins: From the Structure and Function to the Generation of Biotechnological and Therapeutic Tools

“…During the process, the resistive current pulse, the frequency of pore block events, and the time the analyte takes to pass through the pore detection zone are measured. These parameters allow a molecule to be identified according to its distinctive register that is based on its capacity to block the pore [106][107][108].…”