Ideal for species identification of bacteria, fungal, and micro-eukaryotic organisms from uncultured samples of complex communities

The NEXTFLEX® 16S V4 and 18S Amplicon-Seq Kit is designed to prepare libraries that cover both the V4 hypervariable domain of the 16S rRNA genes present in microbial populations and the 18S regions that can identify fungal and micro-eukaryotic organisms in a single library prep. Together with the streamlined workflow and possibility of requesting up to 384 barcodes, this unique kit allows the interrogation of both bacterial and micro-eukaryotic populations within a complex community taking advantage of the high sensitivity of NGS.

Optimized Protocol Offers Low PCR Bias, Fewer Off-target Reads, and Limited Cross-reactivity with Human Samples

The protocol calls for two steps, an initial PCR amplification of the specified target regions using customized PCR primers that target both the 16S V4 and 18S domains, and then a subsequent PCR amplification that adds a sample-specific index. Compared to more traditional sequencing methods, the incorporation of the second PCR step reduces off-target reads typically encountered during amplicon sequencing.

Automated Solution for 16S V4 & 18S rRNA Sequencing

The NEXTFLEX® 16S V4 & 18S Automation kit is a ready-to-go solution for automation on the PerkinElmer® Sciclone G3 NGS/NGSx workstation. The kit includes the library prep reagents with additional overage considerations and single-use, plug-and-play 96-barcode plates to enable a smooth transition for labs wanting a solution for their high-throughput needs. 

Features

• Multiplex up to 384 samples

• Fast and easy joint library prep protocol for both target regions

• Low input – Only 1 ng of input required

• Custom sequencing primers not required

• Automation protocols are available for the PerkinElmer Sciclone® NGS and NGSx Workstations

• Functionally validated on the Illumina® sequencing platforms

Kit Specs

KIT CONTENTS

• NEXTFLEX® PCR Master Mix

• NEXTFLEX® 16S V4 & 18S PCR I Primer Mix

• NEXTFLEX® PCR II Barcoded Primer Mix

• Resuspension Buffer

• Nuclease-free Water

• NEXTFLEX® Cleanup Beads

REQUIRED MATERIALS NOT PROVIDED

• 1 ng – 50 ng high-quality environmental DNA in up to 34 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)

• 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)