Methods for the extraction, storage, amplification and sequencing of DNA from environmental samples

Abstract

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.

Optimal extraction methods for the simultaneous analysis of DNA from diverse organisms and sample types

“…Six different commercially available extraction methods (Table ) were used to isolate DNA from the sample types (Figure ). Most of these methods were selected because they are commonly used to extract DNA from at least one of our target organisms, or from at least one of our environmental sample types (Lear, Dickie, et al., ; Qi et al., ; Zhu, Wu, Dai, Zhang, & Wei, ). The ZymoBIOMICS DNA miniprep (ZB) method, which is relatively new, was included as it claims to be effective for extracting DNA from many different sample types.…”

Arbuscular mycorrhiza and soil organic nitrogen: network of players and interactions

“…Such experiments should carefully be designed to cover all relevant time points in the organic N mineralization and downstream N utilization within the complex microbial communities to seize their temporal dynamics aspect. The unprecedented depth of current NGS methods should be utilized, including less prominently studied, but functionally equally important, microbial groups such as soil protists and nematodes [79,106,135].…”

Towards robust and repeatable sampling methods in eDNA‐based studies

“…Previous reviews of metabarcoding methods provide guidance on statistical replication in sampling (Lennon, ; Prosser, ), methods for processing samples (Lear et al., ), data reporting (Chervitz et al., ; Field et al., ) and bioinformatic analyses (Hiraoka, Yang & Iwasaki, ). However, despite the importance of sample collection in underpinning these later steps, we are not aware of any attempt to critically review methods of sample collection for eDNA studies.…”