Illumina metabarcoding of a soil fungal community

Next generation metabarcoding is becoming an indispensable tool in fungal community ecology. Here we tested Illumina metabarcoding, a method that generates shorter reads but achieves deeper sequencing than 454 metabarcoding approaches. We found that paired-end Illumina MiSeq data cover the full ITS1 in many fungal lineages and are suitable for environmental fungal community assessment. There was substantial read loss during data cleanup (78.6%), which, however, did not impede the analyses, because of the large number of initial sequences (over 4Mio). We observed a high stochasticity in individual PCR reactions. Comparing three repeated sets of PCRs products showed that 58.5% of the total fungal operational taxonomic units (OTUs) found were not recovered by any single set of PCR reactions. Similarly, comparing three annealing temperatures showed that 63.6% of all fungal OTUs were not recovered using any single annealing temperature. These findings suggest that sampling of soil fungal communities is more exhaustive, if we combine repeated PCR products, and PCR products generated at various annealing temperatures. To analyze the above issues we sampled 16 soil cores along a 270 cm transect in a meadow. In total we recovered 3320 fungal OTUs (based on a 95% similarity threshold). Distance decay analysis indicated that community similarity decreased slightly with geographical distance.