Protocols for Investigating the Leaf Mycobiome Using High-Throughput DNA Sequencing

Abstract

High-throughput sequencing of taxon-specific loci, or DNA metabarcoding, has become an invaluable tool for investigating the composition of plant-associated fungal communities and for elucidating plant-fungal interactions. While sequencing fungal communities has become routine, there remain numerous potential sources of systematic error that can introduce biases and compromise metabarcoding data. This chapter presents a protocol for DNA metabarcoding of the leaf mycobiome based on current best practices to minimize errors through careful laboratory practices and validation.

Keywords: Amplicons; Fungal leaf endophytes; Metabarcoding; Mock community; Mycobiome; Next-generation sequencing; Phyllosphere; Phytobiome; Primer bias; Sample bias.