High precision genome sequencing of engineered Gluconobacter oxydans 621H by combining long nanopore and short accurate Illumina reads.

State of the art and novel high-throughput DNA sequencing technologies enable fascinating opportunities and applications in the life sciences including microbial genomics. Short high-quality read data already enable not only microbial genome sequencing, yet can be inadequately to solve problems in genome assemblies and for the analysis of structural variants, especially in engineered microbial cell factories. Single-molecule real-time sequencing technologies generating long reads promise to solve such assembly problems. In our study, we wanted to increase the average read length of long nanopore reads with R9 chemistry and conducted a hybrid approach for the analysis of structural variants to check the genome stability of a recombinant Gluconobacter oxydans 621H strain (IK003.1) engineered for improved growth. Therefore we combined accurate Illumina sequencing technology and low-cost single-molecule nanopore sequencing using the MinION® device from Oxford Nanopore. In our hybrid approach with a modified library protocol we could increase the average size of nanopore 2D reads to about 18.9kb. Combining the long MinION nanopore reads with the high quality short Illumina reads enabled the assembly of the engineered chromosome into a single contig and comprehensive detection and clarification of 7 structural variants including all three known genetically engineered modifications. We found the genome of IK003.1 was stable over 70 generations of strain handling including 28h of process time in a bioreactor. The long read data revealed a novel 1420 bp transposon-flanked and ORF-containing sequence which was hitherto unknown in the G. oxydans 621H reference. Further analysis and genome sequencing showed that this region is already present in G. oxydans 621H wild-type strains. Our data of G. oxydans 621H wild-type DNA from different resources also revealed in 73 annotated coding sequences about 91 uniform nucleotide differences including InDels. Together, our results contribute to an improved high quality genome reference for G. oxydans 621H which is available via ENA accession PRJEB18739.