NCM 2021: Characterisation of genetically modified microorganisms is facilitated by long-read whole-genome sequencing

Genetically modified microorganisms (GMMs) are used in the food industry ‘to obtain higher production efficiency and/or yield of enzymes, vitamins, and additives’; however, they often contain antimicrobial resistance (AMR) genes that can spread to the host. One of Sciensano’s roles in Belgium is to screen food products for contaminants. In this study, Jolien performed an in-depth genomic characterisation of protease-producing GMMs by long-read and short-read whole-genome sequencing. Hybrid assemblies of raw short-read data were compared with raw long-read sequences generated using MinION. After processing, the short- and long-reads were assembled with Unicycler and hybridSPAdes. The long reads were also assembled using Canu. Additional bioinformatics were performed on the long-read sequences. The read length profile showed a distinct peak at 6.6–6.8 kb, which was the length of the recombinant plasmid being studied, suggesting the presence of a high-copy free plasmid with AMR genes. However, a significant fraction of the reads were longer than the plasmid length, indicating that the plasmid had been integrated into the chromosome of the host. Additional filtering was performed to remove reads shorter than the plasmid. The results then showed that most of the reads were concatemers of the plasmid, with up to 11 copies of the transgenic construct. This proved the results from the long-read assembly were not an artefact but a biological feature of the samples. Jolien also confirmed that a limited subset of the reads did support integration into the host genome but, based on the fact that nanopore technology ‘is not prone to sequencing bias’, it was suggested that the integration is unstable and only transiently present in cells.