Impacts of DNA extraction and PCR on DNA metabarcoding estimates of soil biodiversity

Abstract

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.

Non‐specific amplification compromises environmental DNA metabarcoding with COI

“…For many applications repeatability between assays or sampling sites is a requirement, such as the detection of an endangered or invasive species (Grey et al, ). Our results, even considering only the top ten common species, show that detectability can vary between sites with the same genetic marker, and that many more than two PCRs will be required if the rare species are to be detected across multiple PCR and water sample replicates (Dopheide, Xie, Buckley, Drummond, & Newcomb, ).…”

Assessing insect biodiversity with automatic light traps in Brazil: Pearls and pitfalls of metabarcoding samples in preservative ethanol

“…Failure in PCR amplification can be due to several causes, from sample preservation and DNA quality to primer design and thermocycler parameters (Taberlet et al, ). Although a number of DNA extraction approaches are available for environmental samples such as soil (Dopheide, Xie, Buckley, Drummond, & Newcom, ), feces (Rytkönen et al, ), and water (Brannock & Halanych, ), little attention has been given to preservative ethanol. Excepting, Shokralla et al () that successfully amplified COI fragments from a single Lepidoptera larva directly from a preservative medium containing 95% ethanol but also mescal solution, followed by first generation sequencing (but see Ritter, Häggqvist, et al, for a recent method).…”

Promises and pitfalls of using high‐throughput sequencing for diet analysis

“…In contrast, the erroneous sequences produced in PCR products and sequencing increase the rate of false positives (Ficetola et al, ; Flynn, Brown, Chain, MacIsaac, & Cristescu, ). Thus, it is essential that the study design considers methodological procedures that minimize either false negatives by, for example, generating multiple PCR replicates (Dopheide et al, ), or false positives by, for example, only accept sequences present in multiple PCR replicates (Alberdi et al, ), or using statistical approaches to identify erroneous sequences (Frøslev et al, ).…”

“…The DNA extraction method itself may affect the quantity and quality of the extracted DNA (Jedlicka, Sharma, & Almeida, ; Majaneva, Diserud, Eagle, Hajibabaei, & Ekrem, ; Oehm et al, ), with obvious downstream consequences. DNA extraction is especially important when targeting metazoans, compared to prokaryotes and micro‐eukaryotes (Dopheide et al, ). Often eDNA extraction relies on commercially available kits (such as the Qiagen QIAmp DNA Stool Mini Kit and PowerFecal/Soil DNA Kit or the Zymo Xpedition TM Soil/Fecal DNA MiniPrep kit), as they represent fast and standardized solutions.…”