Comparative Evaluation of Commercial DNA Isolation Approaches for Nanopore-only Bacterial Genome Assembly and Plasmid Recovery

Abstract

Abstract The advent of Oxford Nanopore Technologies has undergone significant improvements in terms of sequencing costs, accuracy, and sequencing read lengths, making it a cost-effective, and readily accessible approach for analyzing microbial genomes. A major challenge for bacterial whole genome sequencing by Nanopore technology is the requirement for a higher quality and quantity of high molecular weight DNA compared to short-read sequencing platforms. In this study, using eight pathogenic bacteria, we evaluated the quality, quantity, and fragmented size distribution of extracted DNA obtained from three different commercial DNA extraction kits, and one automated robotic platform. Our results demonstrated significant variation in DNA yield and purity among the extraction kits. The ZymoBIOMICS DNA Miniprep Kit (ZM) provided a higher purity of DNA compared to other kit-based extractions. All kit-based DNA extractions were successfully performed on all twenty-four samples using a single MinION flow cell, with the Nanobind CBB Big DNA kit (NB) yielding the longest raw reads. The Fire Monkey HMW-DNA Extraction Kit (FM) and the automated Roche MagNaPure 96 platform (RO) outperformed in genome assembly, particularly in gram-negative bacteria. A minimum of 30× to 50× read coverages is recommended for genome assembly and plasmid recovery. Our evaluation indicated that the RO platform gave the best overall performance compared to other kits. The RO platform has the additional advantages of full automation and high throughput. However, consideration of upfront costs associated with instruments and reagents is crucial. In conclusion, our study provides valuable guidance for selecting effective kit-based DNA extraction methods for bacterial whole genomes and plasmids recovery.