During metabarcoding of endophytic fungi, many seem to use ITS primer pairs that are not (fully) selective for fungi. We experienced that such primers also amplify the ITS region of an in vitro (non-colonized) clone of the host. In field samples, the plant ITS is the most pronounced. Does this prevent successful metabarcoding on Illumina MiSeq (or similar)? Is a highly specific primer truly necessary? Any hints?
Please see this exceptional review article on fungal barcoding. It just might help.
With the continual improvement in high‐throughput sequencing technology and constant updates to fungal reference databases, the use of amplicon‐based DNA markers as a tool to reveal fungal diversity and composition in various ecosystems has become feasible. However, both the primer selection and the experimental procedure require meticulous verification.
however, with the continual improvement of sequencing technology and constant updating of fungal reference databases, it is necessary to re‐evaluate existing primers and design new sets continuously. Trade‐offs among coverage, specificity, and amplification length will inevitably alter community detection. This will lead to more accurate estimations, especially for soil and other highly diverse ecosystems.
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