Nanopore sequencing of HMW-DNA to compare DNA extraction methods for metagenomics

Background: Oxford Nanopore Technologies (ONT) offers an accessible platform for long-read sequencing, which improves the reconstruction of genomes and helps to resolve complex genomic context, especially in the case of metagenome analysis. To take the best advantage of long-read sequencing, DNA extraction methods must be able to isolate pure high molecular weight (HMW) DNA from complex metagenomics samples, without introducing any bias. New methods released on the market, and protocols developed at the research level, were specifically designed for this application and need to be assessed.Results: In this study, with different bacterial cocktail mixes, analyzed as pure or spiked in synthetic fecal matrix, we evaluated the performances of 6 DNA extraction methods using various cells lysis and purification techniques, from quick and easy, to more time-consuming and gentle protocols, and including a portable method for on-site application. In addition to the comparison of the quality, quantity and purity of the extracted DNA, the performance obtained when doing Nanopore sequencing on a MinION flow cell was also tested. From the results obtained, the Quick-DNA HMW MagBead Kit (Zymo Research) was selected as producing the best yield of pure HMW DNA. Furthermore, this kit allowed an unbiased detection, by Nanopore sequencing, of almost all the species present in a complex mock community.Conclusion: Amongst the 6 tested methods, the Quick-DNA HMW MagBead Kit (Zymo Research) was considered as the most suitable for Nanopore sequencing and would be recommended for metagenomics studies using this technology.