NEXTflex® 18S ITS Amplicon-Seq Kit for Illumina® Platforms

Validated library prep for 18S ITS metagenomics analysis of eukaryotes


Catalog# Product Name Quantity US List Price
NEXTflex® 18S ITS Amplicon-Seq Kit (4 Barcodes)
8 rxns $186 Buy Now
NEXTflex® 18S ITS Amplicon-Seq Kit (12 Barcodes) 24 rxns $436 Buy Now
NEXTflex® 18S ITS Amplicon-Seq Kit (48 Barcodes) 96 rxns $1,570 Buy Now
NEXTflex® 18S ITS Amplicon-Seq Kit (Barcodes 1- 96)
192 rxns $2,981 Buy Now
NEXTflex® 18S ITS Amplicon-Seq Kit (Barcodes 97 - 192)
192 rxns $2,981 Buy Now
NEXTflex® 18S ITS Amplicon-Seq Kit (Barcodes 193 - 288)
192 rxns $2,981 Buy Now
NEXTflex® 18S ITS Amplicon-Seq Kit (Barcodes 289 - 384) 192 rxns
$2,981 Buy Now

NEXTflex™ 18S ITS Amplicon-Seq Kit for Metagenomics
  • Low PCR bias and high on-target reads
  • Fast library prep protocol that covers both ITS1 and ITS2 of 18S rRNA gene
  • Low input – As low as 1 ng of genomic DNA
  • Flexible barcode options– barcoded primers available for multiplexing of up to 384 libraries
  • Compatible with regular Illumina® sequencing primers
  • Automation protocols are now available for the PerkinElmer Sciclone NGS and NGSx Workstation to automate your 18S sequencing
  • Functionally validated on the Illumina® MiSeq® sequencer

The NEXTflex™ 18S ITS Amplicon-Seq Kit is designed to prepare multiplexed amplicon libraries that span both the ITS1 and ITS2 hypervariable Internal Transcribed Spacer regions of eukaryotic 18S ribosomal RNA (rRNA) genes for identification of uncultured fungal and micro-eukaryotic organisms. These libraries are compatible with paired-end sequencing on the Illumina® MiSeq® platform. 


Schematic representing conserved and hyper-variable regions of the 18S rRNA gene

Schematic representing conserved and hyper-variable regions of the 18S rRNA gene


Together, the ease of performing library preparation, extreme multiplexing capabilities obtained with the NEXTflex 18S ITS Amplicon-Seq Kit  and the high sensitivity of NGS provide a convenient and powerful method to detect unique components of eukaryotic communities.

Fast 18S Analysis of Fungi

There are two steps involved in 18S ITS amplicon processing: an initial PCR amplification using customized PCR primers that target the ITS domains, and a subsequent PCR amplification that integrates relevant flow cell binding domains and unique 12 base pair sample indices. A limited number of cleanup steps ensures maximum recovery of amplicons for downstream sequencing. It is highly recommended that sequencing of 18S ITS libraries be performed using the MiSeq® V3 reagent kit (2X300). 

Optimized 18S Amplicon-Seq Protocol Offers Lower PCR Bias and Fewer Off-target Reads

The protocol incorporated in the NEXTflex 18S ITS Amplicon-Seq Kit offers better sequencing results than can be obtained using traditional 18S sequencing protocols. The incorporation of the second PCR step in the protocol for the addition of the sample-specific index reduces the number of off-target reads typically encountered during amplicon sequencing.

Sequencing of the 18S rRNA Gene

The ITS1 and ITS2 regions within the highly conserved 18S rRNA gene are the gold standards used in eukaryotic molecular phylogeny. These ITS regions contains a high level of sequence variation between closely related fungal species and are easy targets to amplify from minimal quantities of DNA due to the high number of copies found per genome, ranging from 10-200. These are excellent markers for detecting both rare species and those in great abundance within complex communities.

Kit Specs

The NEXTflex 18S ITS Amplicon-Seq Kit contains reagents and barcodes to prepare 8, 24, 96 or 192 amplicon-seq libraries from genomic DNA for Illumina® sequencing. The shelf life of all reagents is 12 months when stored properly. All components can be safely stored at -20°C. This kit is shipped on dry ice.

NEXTflex™ 18S ITS Amplicon-Seq Kit Protocol

NEXTflex 18S ITS Amplicon-Seq Illumina Library Prep Protocol



Kit Contents

NEXTflex™ PCR Master Mix

NEXTflex™ 18S ITS PCR I Primer Mix

NEXTflex™ PCR II Barcoded Primer Mix

Resuspension Buffer

Nuclease-free Water


Required Materials Not Provided

1 ng - 50 ng high-quality genomic DNA in up to 36 µL nuclease-free water for each library

96 well PCR Plate Non-skirted (Phenix Research, Cat # MPS-499) or similar

Adhesive PCR Plate Seal (Bio-Rad®, Cat # MSB1001)

Agencourt® AMPure® XP 5 mL (Beckman Coulter® Genomics, Cat # A63880)

Magnetic Stand - 96 (Thermo Fisher Scientific®, Cat # AM10027) or similar


2, 10, 20, 200 and 1000 µL pipettes / multichannel pipettes

Nuclease-free barrier pipette tips


80% Ethanol, freshly prepared (room temperature)

Sequences of NEXTflex 18S ITS Amplicon-Seq Barcoded Primers Indexes - Excel / PDF

Instructions for installing NEXTflex Barcode Indices in Illumina Experiment Manager