Department of Chemistry, Lomonosov Moscow State University, 119991 Moscow, Russia.
Bach Institute of Biochemistry, Federal Research Centre "Fundamentals of Biotechnology" of the Russian Academy of Sciences, 119071 Moscow, Russia.
Int J Mol Sci. 2022 Aug 2;23(15):8569. doi: 10.3390/ijms23158569.
Nanopore sequencing (ONT) is a new and rapidly developing method for determining nucleotide sequences in DNA and RNA. It serves the ability to obtain long reads of thousands of nucleotides without assembly and amplification during sequencing compared to next-generation sequencing. Nanopore sequencing can help for determination of genetic changes leading to antibiotics resistance. This study presents the application of ONT technology in the assembly of an genome characterized by a deletion of the gene and known single-nucleotide variations leading to antibiotic resistance, in the absence of a reference genome. We performed benchmark studies to determine minimum coverage depth to obtain a complete genome, depending on the quality of the ONT data. A comparison of existing programs was carried out. It was shown that the Flye program demonstrates plausible assembly results relative to others (Shasta, Canu, and Necat). The required coverage depth for successful assembly strongly depends on the size of reads. When using high-quality samples with an average read length of 8 Kbp or more, the coverage depth of 30× is sufficient to assemble the complete genome de novo and reliably determine single-nucleotide variations in it. For samples with shorter reads with mean lengths of 2 Kbp, a higher coverage depth of 50× is required. Avoiding of mechanical mixing is obligatory for samples preparation. Nanopore sequencing can be used alone to determine antibiotics-resistant genetic features of bacterial strains.
纳米孔测序（ONT）是一种新的、快速发展的方法，用于确定 DNA 和 RNA 中的核苷酸序列。与下一代测序相比，它在测序过程中无需组装和扩增就能获得数千个核苷酸的长读长。纳米孔测序有助于确定导致抗生素耐药性的遗传变化。本研究介绍了 ONT 技术在组装一个基因组中的应用，该基因组的特征是缺失基因和已知导致抗生素耐药性的单核苷酸变异，而没有参考基因组。我们进行了基准研究，以确定获得完整基因组所需的最小覆盖深度，这取决于 ONT 数据的质量。还比较了现有的程序。结果表明，Flye 程序相对于其他程序（Shasta、Canu 和 Necat）表现出合理的组装结果。成功组装所需的覆盖深度强烈依赖于读取的大小。当使用平均读长为 8 Kbp 或更长的高质量样本时，30×的覆盖深度足以从头组装完整基因组，并可靠地确定其中的单核苷酸变异。对于平均读长为 2 Kbp 的较短读取样本，则需要更高的覆盖深度 50×。对于样本制备，必须避免机械混合。纳米孔测序可单独用于确定细菌菌株的抗生素耐药性遗传特征。
