O0567 Unravelling the complex structure of mobile genetic elements through short- and long-read sequencing

Natacha Couto*2, Monika Chlebowicz1, Ana Carolina Da Cruz Campos1, Erwin G. Raangs1, Alexander W. Friedrich1, John W. A. Rossen2

1University of Groningen, University Medical Center Groningen, Medical Microbiology, Groningen, Netherlands, 2University of Groningen, University Medical Center Groningen, Department of Medical Microbiology, Groningen, The Netherlands

Background: Mobile genetic elements (MGEs) are DNA segments moving within genomes and carriers of antimicrobial resistance and virulence genes. One way to study MGEs is through sequencing, however assembling short-reads is difficult because MGEs contain many repetitive insertion sequences. With long-read sequencing, the full structure of a bacterial genome can be obtained, often with higher sequencing error rates. Here we aim to combine MinION and Illumina data using hybrid-assemblers to unravel the structure of MGEs.

Materials/methods: The DNeasy UltraClean Microbial Kit (Qiagen) was used for DNA extraction of colonies grown overnight at blood agar. Libraries were prepared with the Nextera XT Library Preparation kit (Illumina) and sequenced in a MiSeq (500-cycles, paired-end). For long-read sequencing, samples were barcoded with the Native Barcoding Kit 1D (EXP-NBD103) and libraries were prepared using the Ligation Sequencing Kit 1D (SQK-LSK108). The library was loaded onto an FLO-MIN106 R9.4 flow cell and ran on a MinION device (48 hours). Base calling was performed using Albacore v1.2.2. Data quality was analyzed through Poretools v0.6.0. Hybrid assemblies were performed using Unicycler v0.4.1. Bandage v0.8.1 was used to visualize the assembly graphics. Genes of interest were detected using ABRicate.

Results: One Escherichia coli and one Staphylococcus haemolyticus strain were sequenced. The hybrid assembly produced a complete circular chromosome and several circular plasmids (7 plasmids in E. coli and 2 plasmids in S. haemolyticus). An antimicrobial resistance gene cluster (blaTEM-1B, sul2, strA, strB, tetA) was identified in one IncFII plasmid in E. coli also containing a virulence gene cluster. Several resistance genes (including mecA) were found in the S. haemolyticus chromosome. The SCCmec structure containing the mecA had 99% homology with S. haemolyticus SH32, but the region flanking the mecA was different.

Conclusions: Illumina sequencing is not enough to resolve the complex structure of MGEs, and Nanopore sequencing contains too many sequencing errors to make it reliable. The combination of both methods provides the best solution to understand the intricate sequences surrounding antimicrobial resistance and virulence genes. This is highly desirable to study the dissemination of these important elements.