BackgroundThere is an increasing demand for rapid biodiversity assessment tools that have a broad taxonomic coverage. Here we evaluate a suite of environmental DNA (eDNA) markers coupled with next generation sequencing (NGS) that span the tree of life, comparing them with traditional biodiversity monitoring tools within ten 20×20 meter plots along a 700 meter elevational gradient.ResultsFrom six eDNA datasets (one from each of 16S, 18S, ITS, trnL and two from COI) we identified sequences from 109 NCBI taxonomy-defined phyla or equivalent, ranging from 31 to 60 for a given eDNA marker. Estimates of alpha and gamma diversity were sensitive to the number of sequence reads, whereas beta diversity estimates were less sensitive. The average within-plot beta diversity was lower than between plots for all markers. The soil beta diversity of COI and 18S markers showed the strongest response to the elevational variation of the eDNA markers (COI: r=0.49, p<0.001; 18S: r=0.48, p<0.001). Furthermore pairwise beta diversities for these two markers were strongly correlated with those calculated from traditional vegetation and invertebrate biodiversity measures.ConclusionsUsing a soil-based eDNA approach, we demonstrate that standard phylogenetic markers are capable of recovering sequences from a broad diversity of eukaryotes, in addition to prokaryotes by 16S. The COI and 18S eDNA markers are the best proxies for aboveground biodiversity based on the high correlation between the pairwise beta diversities of these markers and those obtained using traditional methods.Electronic supplementary materialThe online version of this article (doi:10.1186/s13742-015-0086-1) contains supplementary material, which is available to authorized users.

Advances in the sequencing of DNA extracted from media such as soil and water offer huge opportunities for biodiversity monitoring and assessment, particularly where the collection or identification of whole organisms is impractical. However, there are myriad methods for the extraction, storage, amplification and sequencing of DNA from environmental samples. To help overcome potential biases that may impede the effective comparison of biodiversity data collected by different researchers, we propose a standardised set of procedures for use on different taxa and sample media, largely based on recent trends in their use. Our recommendations describe important steps for sample pre-processing and include the use of (a) Qiagen DNeasy PowerSoil ® and PowerMax ® kits for extraction of DNA from soil, sediment, faeces and leaf litter; (b) DNeasy PowerSoil ® for extraction of DNA from plant tissue; (c) DNeasy Blood and Tissue kits for extraction of DNA from animal tissue; (d) DNeasy Blood and Tissue kits for extraction of DNA from macroorganisms in water and ice; and (e) DNeasy PowerWater ® kits for extraction of DNA from microorganisms in water and ice. Based on key parameters, including the specificity and inclusivity of the primers for the target sequence, we recommend the use of the following primer pairs to amplify DNA for analysis by Illumina MiSeq DNA sequencing: (a) 515f and 806RB to target bacterial 16S rRNA genes (including regions V3 and V4); (b) #3 and #5RC to target eukaryote 18S rRNA genes (including regions V7 and V8); (c) #3 and #5RC are also recommended for the routine analysis of protist community DNA; (d) ITS6F and ITS7R to target the chromistan ITS1 internal transcribed spacer region; (e) S2F and S3R to target the ITS2 internal transcribed spacer in terrestrial plants; (f) fITS7 or gITS7, and ITS4 to target the fungal ITS2 region; (g) NS31 and AML2 to target glomeromycota 18S rRNA genes; and (h) mICOIintF and jgHCO2198 to target cytochrome c oxidase subunit I (COI) genes in animals. More research is currently required to confirm primers suitable for the selective amplification of DNA from specific vertebrate taxa such as fish. Combined, these recommendations represent a framework for efficient, comprehensive and robust DNA-based investigations of biodiversity, applicable to most taxa and ecosystems. The adoption of standardised protocols for biodiversity assessment and monitoring using DNA extracted from environmental samples will enable more informative comparisons among datasets, generating significant benefits for ecological science and biosecurity applications.

Soils are ubiquitous and important components of terrestrial ecosystems, richly populated with diverse organisms with important ecological roles. DNA metabarcoding is a promising tool for efficient and taxonomically comprehensive analyses of soil biodiversity, but the outcomes of these analyses are likely affected by basic methodological factors such as sampling and laboratory protocols. We investigated the impacts of DNA extraction methodology and multiple PCRs on DNA metabarcoding biodiversity estimates for multiple taxonomic groups in soil. We applied four DNA extraction methods with different size constraints in parallel to fixed volumes of soil from a pair of forest sites, followed by sets of ten individually indexed PCRs and Illumina sequencing of prokaryote (16S) and eukaryote (18S, fungal 26S, and metazoan cytochrome c oxidase subunit I) barcodes. Methods utilizing larger soil volumes resulted in higher biodiversity estimates for arthropods but not for prokaryotes or microeukaryotes, and improved spatial discrimination of metazoan communities but not prokaryotes. Each DNA extraction method resulted in a biased community composition, and these biases were consistent across sites and amplicons. Some 36%–41% of operational taxonomic units (OTUs) were shared between all four DNA extraction methods and OTUs from many phyla had contrasting abundances from different methods. From 0.2% to 19% of OTUs, accounting for 9.4% to 97% of sequence reads, were amplified in all 10 PCRs from each DNA extraction method and sample. OTUs detected in only a single PCR accounted for 26% to 41% of OTUs but only 0.3% to 2.8% of the sequence reads from each extraction method and sample. The inferred species richness increased strongly with the number of PCRs carried out. We conclude that a consistent DNA extraction method should be used for comparisons between samples, confirm that larger samples of soil (≥15 g) should be used for analyses of metazoan biodiversity than for microbes, and that the number of PCR replicates undertaken strongly affects species richness estimates.