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

Abstract

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 Salmonella enterica genomes within the wild-type strain and strains lacking the PT regulatory gene dndB (ΔdndB) or the PT synthetic gene dndC (ΔdndC). 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 ΔdndB and 25-fold lower in ΔdndC, again consistent with known regulation of the dnd 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.

Modification mapping by nanopore sequencing

“…Moreover, the genomes of several S. aureus jumbo bacteriophages that are presumed to be enriched for uracil-substituted DNA were sequenced on an ONT MinION but produced “sequence data that could not be interpreted” by Korn and others, suggesting that 5-hydroxymethyluracil and/or other deoxyuracil derivatives may produce detectable distortions in nanopore sequencing; the same samples were not sequenceable by standard short read methods until the authors prepared sequencing libraries using the uracil-insensitive polymerase PhusionU ( Korn et al, 2021 ). Finally, the Nookaew laboratory used nanopore sequencing for the detection of phosphorothioate (PS) linkages ( Wadley et al, 2022 ), which occur naturally in prokaryotes and archaea ( Wang et al, 2007 ) and are also commonly used as stabilizing linkages during oligonucleotide synthesis to protect against nuclease digestion ( Vosberg and Eckstein, 1982 ). Using their previously developed ELIGOS software tool, which identifies sites with high base calling error rates via comparative statistical tests between two samples ( Jenjaroenpun et al, 2021 ), they identified sites with statistically significant differences in base calling error rates between PS+ and PS- samples, extracted raw nanopore features with Nanopolish, and analyzed those features to determine PS signatures in Salmonella enterica genomes, noting that ionic current disturbances occur at known sites of PS linkages.…”

“…Using their previously developed ELIGOS software tool, which identifies sites with high base calling error rates via comparative statistical tests between two samples ( Jenjaroenpun et al, 2021 ), they identified sites with statistically significant differences in base calling error rates between PS+ and PS- samples, extracted raw nanopore features with Nanopolish, and analyzed those features to determine PS signatures in Salmonella enterica genomes, noting that ionic current disturbances occur at known sites of PS linkages. This study represents the first use of high throughput nanopore sequencing to detect modifications of the nucleic acid backbone ( Wadley et al, 2022 ).…”

High-throughput sequencing of EcoWI restriction fragments maps the genome-wide landscape of phosphorothioate modification at base resolution

“…A method using optical visualization of PT modifications in single DNA molecules has been developed, however, this method is not high-throughput and doesn’t provide base resolution detection [ 19 ]. More recently, a PT-site detection method using nanopore sequencing was reported for plasmid and bacterial genomic DNA (gDNA) [ 20 ]. A convenient and accurate method for detecting PT modifications is still greatly needed to determine and unravel the unprecedented phosphorothioate epigenomes.…”