Comparison of three DNA extraction kits to establish maximum yield and quality of coral-associated microbial DNA

Abstract

Coral microbiology is an expanding field, yet there is no standard DNA extraction protocol. Although many researchers depend on commercial extraction kits, no specific kit has been optimized for use with coral samples. Both soil and plant DNA extraction kits from MO BIO Laboratories, Inc. have been used by many research groups for this purpose. MO BIO recently replaced their PowerPlant® kit with an improved PowerPlantPro kit, but it was unclear how these changes would affect the kit’s use with coral samples. In order to determine which kit produced the best results, we conducted a comparison between the original PowerPlant kit, the new PowerPlantPro kit, and an alternative kit, PowerSoil, using samples from several different coral genera. The PowerPlantPro kit had the highest DNA yields, but the lack of 16S rRNA gene amplification in many samples suggests that much of the yield may be coral DNA rather than microbial DNA. The most consistent positive amplifications came from the PowerSoil kit. Introduction Due to increased reports of coral diseases and rising interest in the associated microbiomes of animals, coral microbiology has grown and diversified. Researchers seek to characterize the coral holobiont (the totality of the coral animal plus microbial symbionts) in order to explore dispersal, biodiversity, disease, and other aspects of fragile reef ecosystems. Since 2001 molecular techniques such as DNA extraction followed by DNA sequencing have been employed to assess microbial biodiversity and richness in corals (Rohwer and others, 2001). Technical advances, such as pyrosequencing (also known as ‘second-generation’ or ‘next-generation’ sequencing), have driven manufacturers of biological kits and reagents to optimize and specialize their DNA extraction products to improve the overall quality of recoverable data from environmental samples. In spite of the increase in coral microbiology research and publication over the past 12 years (for example, Rohwer and others, 2001; Kellogg, 2004; Bourne and Munn, 2005; Lampert and others, 2008; Kellogg and others, 2009; Lins-de-Barros and others, 2010; Kellogg and others, 2013), we are unaware of any commercial kit optimized for the extraction of microbial DNA from coral samples. Kits designed for other sample types have been used ‘off-label’ for corals, to include soil kits (Rohwer and others, 2001; Penn and others, 2006; Kellogg and others, 2009; Reis and others, 2009; Godwin and others, 2012; Santos and others, 2012), plant kits (Sunagawa and others, 2009; Sato and others, 2010; Sunagawa and others, 2010; Santos and others, 2012; Bayer and others, 2013; Roder and others, 2014) and blood and tissue kits (Sweet and others, 2011). In 2010, Sunagawa and others published a popular protocol based on a modified version of the MO BIO PowerPlant DNA Isolation Kit. Modifications included the addition of chemical lysis agents (lysozyme and protease) and smaller zirconia/silica beads to break open microbial cells during physical lysis. This protocol has been used in a number of recent studies (Sunagawa and others, 2010; Kellogg and others, 2012; Bayer and others, 2013; Kellogg and others, 2013). In early 2013, MO BIO discontinued production of the PowerPlant kit and replaced it with a new product, the PowerPlantPro kit. The