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PENOVA-4210-02
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.
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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.
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).
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.
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.
A NEXTFLEX® automation protocol is now available on the Sciclone® NGS and NGSx workstations to help labs increase their throughput and reduce human errors. Automated versions of the NEXTFLEX® 18S ITS Amplicon-Seq Library Prep kits are available by request.
KIT CONTENTS
REQUIRED MATERIALS NOT PROVIDED
SEQUENCES
BROCHURES
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.
