Nanopore Sequencing for De Novo Bacterial Genome Assembly and Search for Single-Nucleotide Polymorphism

Abstract

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 E. coli genome characterized by a deletion of the tolC 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.