Laszlo Andrew H, Derrington Ian M, Ross Brian C, Brinkerhoff Henry, Adey Andrew, Nova Ian C, Craig Jonathan M, Langford Kyle W, Samson Jenny Mae, Daza Riza, Doering Kenji, Shendure Jay, Gundlach Jens H
Department of Physics, University of Washington, Seattle, Washington, USA.
Department of Genome Sciences, University of Washington, Seattle, Washington, USA.
Nat Biotechnol. 2014 Aug;32(8):829-33. doi: 10.1038/nbt.2950. Epub 2014 Jun 25.
Nanopore sequencing of DNA is a single-molecule technique that may achieve long reads, low cost and high speed with minimal sample preparation and instrumentation. Here, we build on recent progress with respect to nanopore resolution and DNA control to interpret the procession of ion current levels observed during the translocation of DNA through the pore MspA. As approximately four nucleotides affect the ion current of each level, we measured the ion current corresponding to all 256 four-nucleotide combinations (quadromers). This quadromer map is highly predictive of ion current levels of previously unmeasured sequences derived from the bacteriophage phi X 174 genome. Furthermore, we show nanopore sequencing reads of phi X 174 up to 4,500 bases in length, which can be unambiguously aligned to the phi X 174 reference genome, and demonstrate proof-of-concept utility with respect to hybrid genome assembly and polymorphism detection. This work provides a foundation for nanopore sequencing of long, natural DNA strands.
DNA纳米孔测序是一种单分子技术，它可以在最少的样品制备和仪器设备条件下实现长读长、低成本和高速度。在此，我们基于纳米孔分辨率和DNA控制方面的最新进展，来解读DNA通过孔蛋白MspA转运过程中观察到的离子电流水平变化。由于大约四个核苷酸会影响每个水平的离子电流，我们测量了对应于所有256种四核苷酸组合（四联体）的离子电流。这个四联体图谱对于预测源自噬菌体phi X 174基因组的先前未测量序列的离子电流水平具有高度的准确性。此外，我们展示了phi X 174长达4500个碱基的纳米孔测序读数，这些读数可以明确地与phi X 174参考基因组比对，并证明了其在混合基因组组装和多态性检测方面的概念验证实用性。这项工作为长天然DNA链的纳米孔测序奠定了基础。
