# Overview of the Fungal Metagenomic Sequencing Demonstrated Protocol

The Fungal Metagenomic Sequencing Demonstrated Protocol is for the analysis of fungal metagenomic samples, and describes primer sequences and the library preparation workflow. The protocol details a 2-step PCR amplicon workflow for the amplification and analysis of the Internal Transcribed Spacer 1 (ITS1) region.

![](/files/8hOtnpDBKsd4a8O6scen)

**Note:** If using the recommended primers or other sets of primers, dilute an aliquot of each primer to 1 µM with 1X TE pH 8 buffer or nuclease-free water. Pool equal volumes of each primer to create a forward primer pool and a reverse primer pool. Aliquot 5 µl of each primer pool into each library plate well containing sample. There are 8 forward and 7 reverse primers in the Illumina design.

**Resources:**

[Fungal Metagenomic Sequencing Demonstrated Protocol](https://support.illumina.com/downloads/fungal-metagenomic-sequencing-demonstrated-protocol-1000000064940.html)

Uses indexes from the [Nextera XT Index Kit v2](https://www.illumina.com/products/by-type/sequencing-kits/library-prep-kits/nextera-xt-dna.html) sets A - D (Illumina, Catalog No. FC-131-2001, FC-131-2002, FC-131-2003, or FC-131-2004)

[Illumina Learning page for 16S and ITS rRNA Sequencing](https://www.illumina.com/areas-of-interest/microbiology/microbial-sequencing-methods/16s-rrna-sequencing.html)

[Fungal sequencing and classification with the ITS Metagenomics Protocol App Note](https://www.illumina.com/content/dam/illumina-marketing/documents/products/appnotes/its-metagenomics-app-note-1270-2018-001-web.pdf)

BaseSpace Demo Data for ITS from App Note: [Project Data](https://basespace.illumina.com/s/aqqbR3FBY0nL)

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