NEXTFLEX® 18S ITS Amplicon-Seq Kit

Validated library prep for 18S ITS metagenomics analysis of eukaryotes.

Ideal for Gene Identification of Uncultured Fungal & Micro-Eukaryotic Organisms

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® sequencing platforms.

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.

Features

• 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 Workstations to automate your 18S sequencing

• Functionally validated on the Illumina® MiSeq® sequencer

Kit Specs

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

• Thermocycler

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

• Nuclease-free barrier pipette tips

• Vortex

• 80% Ethanol, freshly prepared (room temperature)

Davey, M. P., Norman, L., Sterk, P., Huete-Ortega, M., Bunbury, F., Loh, B. K., . . . Smith, A. G. (2019). Snow algae communities in Antarctica – metabolic and taxonomic composition. New Phytologist. doi:10.1111/nph.15701