Saturated cell lysing is critical for high sensitivity microbiome analysis

Abstract

Introductory paragraphFor robust DNA-based gut microbiome analysis, all cells in the stool samples need to be lysed. However, no standards have been developed to evaluate a DNA extraction protocol’s capability of lysing all cells and its sensitivity on detecting microbial structural differences among samples. In this study, we incrementally increased the intensity of mechanical lysis and integrated lysozyme pretreatment to Protocol Q (PQ), which was recommended as the best from 21 protocols1. A new protocol (LPQ) was optimized when DNA yield, Gram-positive bacteria ratio, and beta diversity all reached to a plateau with no further significant changes, indicating the achievement of saturated lysing. LPQ detected significant differences among three groups of fiber-treated human stool samples and identified 64 responsive ASVs, while a commercial kit failed to detect any significant treatment effects and PQ only detected 17 responsive ASVs. Therefore, saturated lysing as defined in this study should be adopted for evaluating microbiome DNA extraction protocols.