Implementation of a flexible Oxford Nanopore sequencing platform for microbial genomics

Oxford Nanopore sequencing technology is slowly revolutionising the entire microbiology field. Its ease of use and cost effective approaches coupled with long reads sequencing represent an essential and powerful tool. In the present thesis I have implemented a sequencing platform based on the Oxford Nanopore technology, a flexible system suitable for both research and diagnostic fields. The first part of my work was dedicated to the optimization of a DNA extraction protocol capable of isolating high molecular weight (HMW) genomic DNA. In fact, Nanopore sequencing readouts are highly influenced by both the quality and the integrity of the genomic DNA. An enzymatic lysis based extraction protocol was optimized, recovering HMW DNA from two strains of Streptococcus mitis and generating multiple ultra long reads (i.e. >100 Kb in length), making it possible to achieve complete genome assemblies. As the extraction protocol was mainly optimized for Gram-positive bacteria, it is also suitable to lyse the thinner cell wall of Gram-negatives. Oxford Nanopore Whole Genome Sequencing (WGS) approaches have enabled the complete genome sequencing and assembly of 16 Enterococcus faecalis isolates from clinical dental samples. Sequencing data provided enough information enabling i) population studies, defining genomic clusters based on isolates homologies; ii) bacterial profiling, assessing antimicrobial resistance genes and virulence traits; iii) comparative analysis, identifying genomic rearrangements and homologies based on synteny blocks. Finally, the platform was used for the monitoring of the ongoing SARS-CoV-2 pandemic. We have proposed a 900 bp amplicon sequencing protocol, adapted from the ARTIC sequencing protocol (https://artic.network/), enabling a near-complete genome assembly of SARS-CoV-2 strains, helping in the detection of nucleotide changes and monitoring the circulating viral lineages. In conclusion, the Oxford Nanopore sequencing platform can bring several improvements in the microbiology field, allowing i) complete genome assembly, ii) rapid microbial profiling, and iii) helping in the monitoring of local or global outbreaks.