An optimized protocol for high-throughput amplicon-based microbiome profiling

Here we describe an optimized protocol for amplicon-based microbiome pro ling by next-generation sequencing \(NGS).The protocol uses a modular, two-step PCR process that provides versatility as a common set of indexing primers can be paired with many sets of marker gene speci c primers.We describe primer sets that have been used to prepare libraries for variable regions V1-V3, V3-V4 V3-V5, V4, V4-V6, and V5-V6 of the 16S ribosomal RNA \(rRNA) gene, as well as for variable region V9 of the 18S rRNA gene and the ITS1 and ITS2 regions.This protocol is designed for high-throughput and incorporates the best practices for ampliconbased NGS library preparation that we have previously described.We have demonstrated that these best practices substantially increase accuracy compared to existing methods and also, importantly, minimize the dropout of taxa that have mismatches to the ampli cation primers.