Optimisation of a DNA extraction protocol for improving the bacterial and fungal classification based on Nanopore sequencing

Ribosomal RNA gene amplicon sequencing is commonly used to evaluate microbiome profiles in health and disease and document the impact of interventional treatments. Nanopore sequencing is attractive since it can provide greater classification at the species level. However, optimised protocols to target marker genes for bacterial and fungal profiling are needed. To achieve an increased taxonomic resolution, we developed extraction and full-length amplicon PCR-based approaches using Nanopore sequencing. Three lysis conditions were applied to a mock microbial community, including known bacterial and fungal species; the ZymoBIOMICSTM lysis buffer (ML) alone, incorporating bead-beating (MLB), or bead-beating plus MetaPolyzyme enzymatic treatment (MLBE). Profiling of bacterial comparison to reference data, MLB had more statistically different bacterial phyla and genera than the others. Fungal profiling, MLB had a significant increase of Ascomycota and a decline of Basidiomycota, subsequently failing to detect Malassezia and Cryptococcus. Also, the principal coordinates analysis (PCoA) plot by the Bray-Curtis metric showed a significant difference among groups for bacterial (p = 0.033) and fungal (p = 0.012) profiles, highlighting the importance of understanding the biases present in pretreatment. Overall, the microbial profiling and diversity analysis revealed that ML and MLBE have more similarity than MLB for both bacteria and fungi; therefore, using this specific pipeline, bead-beating is not recommended for whole gene amplicon sequencing. However, ML alone was suggested as an optimal approach considering DNA yield, taxonomic classification, reagent cost and hands-on time. This could be an initial proof-of-concept study for simultaneous human bacterial and fungal microbiome studies.