Choice of bead-beater instrument can result in significant differences in the outcome of host-associated microbiome studies

Abstract

Background: Accurate assessment of the abundance and composition of microbial assemblages in a complex environmental sample depends on the successful lysis of microbial cells, for which bead-beating is typically used. In this study, we compared two commonly used bead beaters, FastPrep and the Bead Ruptor Elite, for their ability to lyse the eastern-oyster-associated bacterial communities over three different time points. Methods:  Genomic DNA was extracted from homogenized oyster samples using two different lysis equipment: the MSP FastPrep and the Bead Ruptor Elite. The V4-V5 variable regions of microbial small subunit ribosomal RNA (16S rRNA) genes were PCR-amplified and sequenced using Illumina Miseq, obtained sequences were bioinformatically processed using QIIME2 and the MicrobiomeAnalyst pipeline. Results: We found that the oyster samples were mostly populated by Proteobacteria phyla, regardless of lysis method. Additionally, the samples isolated by the FastPrep lysis method also harbored Firmicutes and Bacteroidota, which were not identified in the samples treated with the Bead Ruptor Elite lysis equipment. Differences were more obvious at the genus level, such that Delftia genus dominated at 80-85% when the lysis was performed using the FastPrep method. Conversely, 80-90% of the microbial abundances in the Bead Ruptor Elite-treated samples belonged to Burkholderia spp. Diversity and evenness estimates revealed that the FastPrep-treated samples were 40% more diverse and 70% more evenly distributed relative to the Bead Ruptor Elite method. Furthermore, principal component analysis (PCA) led to a distinct separation of the bacterial communities retrieved from the two lysis methods. Conclusions: Overall, this study shows that two different lysis protocols can yield significantly different microbial taxa from the same sample; thus, researchers need to be cognizant of DNA extraction process being followed for metagenomics studies, especially those that involve host tissues containing high amounts of mucous and other PCR inhibitory materials.