华盛顿大学Jens H. Gundlach团队近日取得一项新成果。他们的研究认为，利用时变跨膜电压可以提高纳米孔DNA测序的准确度。 2019年6月出版的《Nature Biotechnology》发表了这项成果。

实验中，研究人员采取固定频率为200Hz，通过调节驱动电压从100-200mv，改变DNA链穿过耻垢分枝杆菌孔蛋白A纳米孔(MspA)的方式。DNA解旋酶以不连续的步骤使DNA移动通过纳米孔，并且可变电压在这些步骤之间连续移动DNA，可变电压产生的电信号能克服碱基识别的主要错误模式。该课题组人员发现,当使用可变电压时单通道碱基识别准确度从使用恒定电压时的62.7 ± 0.5%提高到了79.3 ± 0.3%。该调压测序方式是对其他提高纳米孔测序的准确性方法的补充,并且可以与任何酶驱动的纳米孔测序装置相结合。

据悉，纳米孔DNA测序受制于较低的碱基识别准确度。迄今为止，提升碱基识别准确性依靠的是特定的碱基识别算法、不同的纳米孔和酶促分子马达,或采用生物化学方法对DNA分子进行重读。阻碍测序准确性的主要的误差形式有两种：酶差错和序列信号不可分辨。

附：英文原文

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

Author: Matthew T. Noakes, Henry Brinkerhoff, Andrew H. Laszlo, Ian M. Derrington, Kyle W. Langford, Jonathan W. Mount, Jasmine L. Bowman, Katherine S. Baker, Kenji M. Doering, Benjamin I. Tickman, Jens H. Gundlach

Issue&Volume: Volume 37 Issue 6, June 2019

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 from 100 to 200mV, with a frequency of 200Hz, across a Mycobacterium smegmatis porin A (MspA) nanopore, thus changing how the DNA strand moves through the nanopore. A DNA helicase moves the DNA through the nanopore in discrete steps, and the variable voltage moves the DNA continuously between these steps. The electronic signal produced with variable voltage is used to overcome the primary error modes in base calling. We found that single-passage de novo base-calling accuracy of 62.70.5% with a constant driving voltage improves to 79.30.3% with a variable driving voltage. The variable-voltage sequencing mode is complementary to other methods to boost the accuracy of nanopore sequencing and could be incorporated into any enzyme-actuated nanopore sequencing device.

DOI: 10.1038/s41587-019-0096-0

Source:https://www.nature.com/articles/s41587-019-0096-0