Evaluation of DNA extraction kits for long-read shotgun metagenomics using Oxford Nanopore sequencing for rapid taxonomic and antimicrobial resistance detection

Abstract Background: During a bacterial infection, the detection of Antimicrobial Resistance (AMR) is critical, but slow due to culture-based approaches for clinical and screening samples. Culture-based phenotypic AMR detection and confirmation require up to 72 hours(h) or even weeks for slow-growing bacteria. Direct shotgun metagenomics by long-read sequencing using Oxford Nanopore Technologies (ONT) may allow us to reduce the time for bacterial species and AMR gene identification. However, screening swabs for metagenomics is complex due to the range of Gram-negative and -positive bacteria, diverse AMR genes, and host DNA present in the samples. We aimed to compare the performance of different DNA extraction protocols with ONT for reliable species and AMR identification using a shotgun long-read metagenomic approach. Materials and Methods: We included three different sample types: ZymoBIOMICS Microbial Community Standard, inhouse ESKAPE pathogens Mock (covering Enterococcus faecium, Staphylococcus aureus, Klebsiella pneumoniae, Acinetobacter baumannii, Pseudomonas aeruginosa, and Escherichia coli), and leftover anonymized rectal swabs. We processed all sample types with four different DNA extraction kits utilizing different lysis (enzymatic vs. mechanical) and purification (spin-column vs. magnetic beads) methods. We used kits from Qiagen (QIAamp DNA Mini and QIAamp PowerFecal Pro DNA) and Promega (Maxwell RSC Cultured Cells and Maxwell RSC Buccal Swab DNA). We used the Rapid Barcoding Kit (RBK004) for library preparation and sequenced the libraries on the GridION with R9.4.1 flow cells. Results: We identified all bacterial species present in the Zymo Mock Community (8/8) and ESKAPE Mock (6/6) with mechanical lysis at read and assembly levels. Enzymatic lysis failed to retrieve Gram-positive species (Staphylococcus aureus and Enterococcus faecium) from the ESKAPE Mock on the assembly level. We detected the AMR genes from Gram-negative and -positive species in the ESKAPE Mock with a mechanical lysis-based DNA extraction kit (QIAamp PowerFecal Pro DNA kit) on reads and assembly levels within an average of 2.7h of sequencing. Conclusion: Long-read metagenomics with ONT may reduce the turnaround time (TAT) in screening for AMR genes. At this moment, the QIAamp PowerFecal Pro DNA kit for DNA extraction along with the Rapid Barcoding Kit for the ONT sequencing seemed to capture the best taxonomy and AMR identification for our specific use case.