Soil sampling and isolation of extracellular DNA from large amount of starting material suitable for metabarcoding studies PIERRE TABERLET, SOPHIE M. PRUD’HOMME, ETIENNE CAMPIONE, JULIEN ROY, CHRISTIAN MIQUEL, WASIM SHEHZAD, LUDOVIC GIELLY, DELPHINE RIOUX, PHILIPPE CHOLER, JEAN-CHRISTOPHE CLE ´ MENT, CHRISTELLE MELODELIMA, FRANC ¸OIS POMPANON and ERIC COISSAC Laboratoire d’Ecologie Alpine, CNRS UMR 5553, Universite ´ Joseph Fourier, BP 53, F-38041 Grenoble Cedex 9, France Abstract DNA metabarcoding refers to the DNA-based identification of multiple species from a single complex and degraded environmental sample. We developed new sampling and extraction protocols suitable for DNA metabarcoding analyses targeting soil extracellular DNA. The proposed sampling protocol has been designed to reduce, as much as possible, the influence of local heterogeneity by processing a large amount of soil resulting from the mixing of many different cores. The DNA extraction is based on the use of saturated phosphate buffer. The sampling and extraction protocols were validated first by analysing plant DNA from a set of 12 plots corresponding to four plant communities in alpine meadows, and, second, by conducting pilot experiments on fungi and earthworms. The results of the validation experiments clearly demonstrated that sound biological information can be retrieved when following these sampling and extraction procedures. Such a protocol can be implemented at any time of the year without any preliminary knowledge of specific types of organisms during the sampling. It offers the opportunity to analyse all groups of organisms using a single sampling ⁄ extraction procedure and opens the possibility to fully standardize biodiversity surveys. Keywords: DNA extraction, DNA metabarcoding, extracellular DNA, soil Received 20 July 2011; revision received 5 September 2011; accepted 9 September 2011 Introduction DNA barcoding, the use of a standardized DNA sequence to identify species, is becoming a popular solution for taxonomic identification of individual spec- imens. For both animals and plants, the standardized DNA fragments are longer than 500 bp (Hebert et al. 2003; Hollingsworth et al. 2009). Besides the identifica- tion of individual specimens, the barcoding concept has been extended to the identification of multiple taxa based on a single experiment. Such an approach is mainly implemented for bacteria (e.g. Sogin et al. 2006), fungi (e.g. Zinger et al. 2009a), nematodes (e.g. Porazinska et al. 2009), herbivore and carnivore diet studies (e.g. Valentini et al. 2009; Kowalczyk et al. 2011; Shehzad et al. 2012), and solving ecological ques- tions concerning river benthos (Hajibabaei et al. 2011), plants Yoccoz et al. 2012, L. Gielly, et al., in revision) and earthworms (Bienert et al. 2012). The approach consisting of identifying multiple spe- cies, in a single experiment, using complex and degraded environmental samples can be termed ‘DNA metabarcoding’. As soil contains DNA remains from many organisms, it is tempting to use soil DNA to assess biodiversity using a DNA metabarcoding approach. Total soil DNA includes cellular DNA origi- nating from living cells or from living multicellular organisms, and extracellular DNA (Levy-Booth et al. 2007; Pietramellara et al. 2009). Usually, extracellular DNA originates from cell lysis and represents a signifi- cant proportion of total soil DNA (Pietramellara et al. Correspondence: Pierre Taberlet, Fax: +33(0)4 76 51 42 79; E-mail: pierre.taberlet@ujf-grenoble.fr Ó 2012 Blackwell Publishing Ltd Molecular Ecology (2012) 21, 1816–1820 doi: 10.1111/j.1365-294X.2011.05317.x