DNA extraction protocols cause differences in 16S rRNA amplicon sequencing efficiency but not in community profile composition or structure

MicrobiologyOpen

Benjamin E. R. Rubin²  Jon G. Sanders³  Jarrad Hampton-Marcell¹  Sarah M. Owens¹  Jack A. Gilbert¹ 

[1] Institute of Genomic and Systems Biology, Argonne National Laboratory, Lemont, Illinois;Committee on Evolutionary Biology, University of Chicago, Chicago, Illinois;Department of Organismic and Evolutionary Biology, Harvard University, Cambridge, Massachusetts

关键词: 16S rRNA;    ants;    DNA extraction;    Earth Microbiome Project;    host‐associated bacteria;    insects;    microbiome;   

DOI  :  10.1002/mbo3.216

来源: Wiley

【 摘 要 】

Abstract

The recent development of methods applying next-generation sequencing to microbial community characterization has led to the proliferation of these studies in a wide variety of sample types. Yet, variation in the physical properties of environmental samples demands that optimal DNA extraction techniques be explored for each new environment. The microbiota associated with many species of insects offer an extraction challenge as they are frequently surrounded by an armored exoskeleton, inhibiting disruption of the tissues within. In this study, we examine the efficacy of several commonly used protocols for extracting bacterial DNA from ants. While bacterial community composition recovered using Illumina 16S rRNA amplicon sequencing was not detectably biased by any method, the quantity of bacterial DNA varied drastically, reducing the number of samples that could be amplified and sequenced. These results indicate that the concentration necessary for dependable sequencing is around 10,000 copies of target DNA per microliter. Exoskeletal pulverization and tissue digestion increased the reliability of extractions, suggesting that these steps should be included in any study of insect-associated microorganisms that relies on obtaining microbial DNA from intact body segments. Although laboratory and analysis techniques should be standardized across diverse sample types as much as possible, minimal modifications such as these will increase the number of environments in which bacterial communities can be successfully studied.

【 授权许可】

CC BY   
© 2014 The Authors. MicrobiologyOpen published by John Wiley & Sons Ltd.

Creative Commons Attribution License, which permits use, distribution and reproduction in any medium, provided the original work is properly cited.