Background InformationHigh throughput amplicon sequencing is an extremely sensitive technique. The lab environment is full of airborne bacterial and fungal cells as well as bacterial and fungal genomic DNA from cultures, and so extreme care must be taken to keep reactions uncontaminated and in preventing cross-contamination.This protocol was modified from the IBEST Geomics Core at University of Idaho.\u00a0 It utilizes a 2-step PCR, where the first PCR amplifies the specific region of interest (e.g., ITS or 16S V4), and the second PCR adds adapters and barcodes to these amplicons. The target specific primers include (in addition to the standard primer sequence) the universal sequences CS1 and CS2, which provide the bases used in the second PCR to extend the amplicons with Illumina adapters and sample-identification barcodes.Illumina adapters and sample-identification barcodes are not included in the region-specific primers used in PCR1. This allows for maximum flexibility in choosing which primers to use and provides the ability to swap out targets, or include multiple targets in the same sequencing reaction, without needing to purchase a large number of barcoded target specific primers.Barcodes are included in both adapters, therefor a pair of barcodes is used to uniquely identify each sample during bioinformatic processing. This allows us to use 32 barcode pairs to uniquely identify 1024 samples (32 x 32).Phase-shifted primers are used to introduce 1, 2, and\/or 3 additional bases to allow for the maximum amount of \u201csequence diversity\u201d in the first 4 bases. This is important because of how the Illumina sequencers detect clusters on the flowcell during sequencing. If sequences are too similar (as is often the case with amplicons), then sequencing yields will be much lower than expected. 2-bp linkers and phase-shifting bases were chosen to have low identity between primers and the target sequence.Tagged primers were ordered from IDT (standard desalting; not HPLC-purified), re-suspended at 100 \u03bcM using a new bottle of molecular grade water in the PCR-free hood, and mixed in equimolar concentration (250 \u03bcl each).\u00a0 Working stocks were diluted to 50 \u03bcM for PCR1.