Effects of DNA preservation solution and DNA extraction methods on microbial community profiling of soil

Paul Iturbe-Espinoza,Martin Braster,Rob J. M. van Spanning,Bernd W. Brandt,David M. Brown,Matthijs Bonte

doi:10.1007/s12223-021-00866-0

Abstract

Microbial community profiling using high-throughput sequencing relies in part on the preservation of the DNA and the effectiveness of the DNA extraction method. This study aimed at understanding to what extent these parameters affect the profiling. We obtained samples treated with and without a preservation solution. Also, we compared DNA extraction kits from Qiagen and Zymo-Research. The types of samples were defined strains, both as single species and mixtures, as well as undefined indigenous microbial communities from soil. We show that the use of a preservation solution resulted in substantial changes in the 16S rRNA gene profiles either due to an overrepresentation of Gram-positive bacteria or to an underrepresentation of Gram-negative bacteria. In addition, 16S rRNA gene profiles were substantially different depending on the type of kit that was used for extraction. The kit from Zymo extracted DNA from different types of bacteria in roughly equal amounts. In contrast, the kit from Qiagen preferentially extracted DNA from Gram-negative bacteria while DNA from Gram-positive bacteria was extracted less effectively. These differences in kit performance strongly influenced the interpretation of our microbial ecology studies.

Highlights

In recent years, high-throughput sequencing, such as the 16S rRNA gene amplicon sequencing, has contributed to a detailed understanding of the composition of microbial communities (Sinclair et al 2015)
The effect of the DNA preservation solution on the DNA extraction efficiency was studied with four different soil samples from a previous mesocosm experiment (Brown et al 2017)
The relative abundances of Gram-positive genera, such as Bacillus, Paenibacillus, Lysinibacillus, and unclassified members of the family Bacillaceae and Planococcaceae, increased for the samples treated with DNA preservation solution as compared to the ones from samples delivered without preservation solution (Fig. 1)

Summary

Introduction

High-throughput sequencing, such as the 16S rRNA gene amplicon sequencing, has contributed to a detailed understanding of the composition of microbial communities (Sinclair et al 2015). To minimize bias in the quantification of microbial communities due to the breakdown of cells or DNA, it is recommended to extract the DNA immediately upon soil collection If this is not possible, preserving the soil samples may be achieved via snap freezing in liquid nitrogen or storing at − 80 °C though these techniques are not always possible in remote areas (Sessitsch et al 2002; Straube and Juen 2013; Weißbecker et al 2017). Based on the handbook of LifeGuard® Soil Preservation, the preservative does not destroy the cells, but protects the viability of bacteria in soil while keeping them in stasis. The effects of these solutions on the DNA extraction efficiency, are not currently well understood, nor is it understood whether

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