Oxford nanopore technology R10 improved assembly metrics of Borrelia plasmids compared to R9

In this project, we compared whole-genome Borrelia assemblies using reads produced by Oxford Nanopore Technology (ONT) with R9 and R10 chemistry and flow cells to investigate potential improvements between versions. For this, we sequenced two B. garinii isolates and one B. tillae isolate using ONT (R9 and R10) and reconstructed the genome using three different assemblers (Canu, Flye, Shasta). The generated consensus sequences were compared to references (Illumina for B. garinii, PacBio for B. tillae). A general improvement of genome assemblies between R9 and R10 ONT versions was visible for both B. garinii isolates and B. tillae. This improvement was observed for all assemblers and can be clearly seen in the completeness and the decrease in the number of gaps. Using ONT, it was possible to generate at least one contig for each expected plasmid for both isolates of B. garinii, and even close some gaps that were present in Illumina references (Illumina-REF). Additionally, previously undefined contigs of the Illumina-REF assembly could be identified as parts of plasmid lp28-2, lp28-3, and lp32-10 based on the ONT assemblies. For the more complex genome of B. tillae, ONT was only able to generate complete linear plasmids, but all circular plasmids containing sequence stretches of high similarity described in a PacBio reference assembly resulted in a “wrong fusion”. For all samples the use of different assemblers (Canu and Flye) was necessary to generate a whole genome sequence, with choice of assembler used for the final consensus sequence of individual genome elements varying between Borrelia species. ONT R10 chemistry and flow cells greatly improved assembly of B. garinii genomes lacking complex circular plasmids and even fully resolved incomplete linear plasmids (gap closure) generated using Illumina sequencing. However, circular plasmids of B. tillae having sequence stretches of high similarity were not properly assembled. This implies that additional sequencing methods are needed when dealing with complex bacterial genomes.