Metabarcoding of a mock community of soil invertebrates: DNA extraction, false-positives, and data filtration
Metabarcoding is a powerful tool widely used to analyze soil communities but methodological difficulties can introduce biases, resulting in inaccurate estimation of species diversity. We evaluated five DNA extraction methods, two PCR programs, and three post-bioinformatic filtering techniques (multiple databases, filtering based on the level of similarity and on the number of reads obtained) using a mock soil community of invertebrates containing 24 species, 23 genera, and 15 families of arthropods and annelids. The choice of DNA extraction kit significantly influenced the accuracy of the metabarcoding results. Kits developed for the isolation of DNA from soil and alkaline lysis without purification showed worse results compared to those designed for isolating DNA from animal tissues. Increasing the annealing temperature of primers resulted in an increased number of false positives. On the contrary, the use of post-bioinformatic filtering dramatically increased the proportion of true positives, reaching 100% correctness for families, more than 90 % for genera and more than 75% for species forming the mock community. Thus, an acceptable level of accuracy may be achieved in field surveys with relatively little time and effort.