Nanopore Sequencing for Detection and Characterization of Phosphorothioate Modifications in Native DNA Sequences

Abstract

<jats:p>Bacterial DNA is subject to various modifications involved in gene regulation and defense against bacteriophage attacks. Phosphorothioate (PT) modifications are protective modifications in which the non-bridging oxygen in the DNA phosphate backbone is replaced with a sulfur atom. Here, we expand third-generation sequencing techniques to allow for the sequence-specific mapping of DNA modifications by demonstrating the application of Oxford Nanopore Technologies (ONT) and the ELIGOS software package for site-specific detection and characterization of PT modifications. The ONT/ELIGOS platform accurately detected PT modifications in a plasmid carrying synthetic PT modifications. Subsequently, studies were extended to the genome-wide mapping of PT modifications in the <jats:italic>Salmonella enterica</jats:italic> genomes within the wild-type strain and strains lacking the PT regulatory gene <jats:italic>dndB</jats:italic> (<jats:italic>ΔdndB</jats:italic>) or the PT synthetic gene <jats:italic>dndC</jats:italic> (<jats:italic>ΔdndC</jats:italic>). PT site-specific signatures were observed in the established motifs of GAAC/GTTC. The PT site locations were in close agreement with PT sites previously identified using the Nick-seq technique. Compared to the wild-type strain, the number of PT modifications are 1.8-fold higher in <jats:italic>ΔdndB</jats:italic> and 25-fold lower in <jats:italic>ΔdndC</jats:italic>, again consistent with known regulation of the <jats:italic>dnd</jats:italic> operon. These results demonstrate the suitability of the ONT platform for accurate detection and identification of the unusual PT backbone modifications in native genome sequences.</jats:p>