Hidden components of microbiome diversity revealed by cell wall lysis with Adaptive Focused Acoustics

BioRxiv : the Preprint Server for Biology
G. F. WallaceShurjo K Sen

Abstract

Within the rapidly evolving field of microbiome sequencing, a primary need exists for experimentally capturing microbiota in a manner as close as possible to their in vivo composition. During microbiome profiling, the first step necessarily involves lysis of the cell wall, releasing nucleic acids for next-generation sequencing. Microbial cell wall thicknesses can vary between 5nm to 80nm; while some species are quite easy to lyse, others are particularly resistant to lysis. Despite this, current chemical/mechanical lysis protocols ignore the possibility that species with different cell wall thicknesses are lysed at differential rates. This creates noise in species compositions and possibly skews current microbiome results in ways that are not currently understood. To develop a cell wall thickness-agnostic lysis protocol, we used Adaptive Focused Acoustics (AFA), a tunable acoustic methodology for processing of biological samples. Using identical aliquots of mouse stool homogenate as the lysis substrate, we compared AFA with chemical/mechanical lysis methodology routinely used in microbiome studies and found that AFA-mediated lysis substantially increases both microbial DNA yield as well as alpha and beta diversity. By starting ...Continue Reading

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